TITLE OF THE INVENTION

POLYPEPTIDES AND POLYNUCLEOTIDES, AND USES THEREOF FOR TREATMENT OF IMMUNE RELATED DISORDERS AND CANCER

FIELD OF THE INVENTION

This invention relates to LY6G6F, VSIG10, TMEM25 and LSR proteins, which are suitable targets for immunotherapy, treatment of cancer, infectious disorders, and/or immune related disorders, and drug development, as well as soluble molecules and conjugates thereof, and antibodies against such.

BACKGROUND OF THE INVENTION

Naive T cells must receive two independent signals from antigen-presenting cells (APC) in order to become productively activated. The first, Signal 1, is antigen-specific and occurs when T cell antigen receptors encounter the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (Signal 2) which is delivered through a T cell costimulatory molecule that engages its APC-expressed ligand. This second signal could be either stimulatory (positive costimulation) or inhibitory (negative costimulation or coinhibition). In the absence of a costimulatory signal, or in the presence of a coinhibitory signal, T-cell activation is impaired or aborted, which may lead to a state of antigen-specific unresponsiveness (known as T-cell anergy), or may result in T-cell apoptotic death.

Costimulatory molecule pairs usually consist of ligands expressed on APCs and their cognate receptors expressed on T cells. The prototype ligand/receptor pairs of costimulatory molecules are B7/CD28 and CD40/CD40L. The B7 family consists of structurally related, cell-surface protein ligands, which may provide stimulatory or inhibitory input to an immune response. Members of the B7 family are structurally related, with the extracellular domain containing at least one variable or constant immunoglobulin domain.

Both positive and negative costimulatory signals play critical roles in the regulation of cell-mediated immune responses, and molecules that mediate these signals have proven to be effective targets for immunomodulation. Based on this knowledge, several therapeutic approaches that involve targeting of costimulatory molecules have been developed, and were shown to be useful for prevention and treatment of cancer by turning on, or preventing the turning off, of immune responses in cancer patients and for prevention and treatment of autoimmune diseases and inflammatory diseases, as well as rejection of allogenic transplantation, each by turning off uncontrolled immune responses, or by induction of "off signal" by negative costimulation (or coinhibition) in subjects with these pathological conditions.

Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases, and transplant rejection. Therapeutic strategies include blocking of costimulation using monoclonal antibodies to the ligand or to the receptor of a costimulatory pair, or using soluble fusion proteins composed of the costimulatory receptor that may bind and block its appropriate ligand. Another approach is induction of co-inhibition using soluble fusion protein of an inhibitory ligand. These approaches rely, at least partially, on the eventual deletion of auto- or allo-reactive T cells (which are responsible for the pathogenic processes in autoimmune diseases or transplantation, respectively), presumably because in the absence of costimulation (which induces cell survival genes) T cells become highly susceptible to induction of apoptosis. Thus, novel agents that are capable of modulating costimulatory signals, without compromising the immune system's ability to defend against pathogens, are highly advantageous for treatment and prevention of such pathological conditions.

Costimulatory pathways play an important role in tumor development. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation through inhibition of co-stimmulatory factors in the B7-CD28 and TNF families, as well as by attracting regulatory T cells, which inhibit anti-tumor T cell responses (see Wang (2006) Immune Suppression by Tumor Specific CD4+ Regulatory T cells in Cancer. Semin. Cancer. Biol. 16:73-79; Greenwald, et al. (2005) The B7 Family Revisited. Ann. Rev. Immunol. 23:515-48; Watts (2005) TNF/TNFR Family Members in Co-stimulation of T Cell Responses Ann. Rev. Immunol. 23:23-68; Sadum, et al. (2007) Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Targets for Cancer Immunotherapy. Clin. Cane. Res. 13(13): 4016-4025). Such tumor expressed co-stimulatory molecules have become attractive cancer biomarkers and may serve as tumor-associated antigens (TAAs). Furthermore, costimulatory pathways have been identified as immunologic checkpoints that attenuate T cell dependent immune responses, both at the level of initiation and effector function within tumor metastases. As engineered cancer vaccines continue to improve, it is becoming clear that such

immunologic checkpoints are a major barrier to the vaccines' ability to induce therapeutic anti-tumor responses. In that regard, costimulatory molecules can serve as adjuvants for active (vaccination) and passive (antibody-mediated) cancer immunotherapy, providing strategies to thwart immune tolerance and stimulate the immune system.

In addition, such agents could be of use in other types of cancer immunotherapy, such as adoptive immunotherapy, in which tumor-specific T cell populations are expanded and directed to attack and kill tumor cells. Agents capable of augmenting such anti-tumor response have great therapeutic potential and may be of value in the attempt to overcome the obstacles to tumor immunotherapy. Recently, novel agents that modulate several costimulatory pathways were indeed introduced to the clinic as cancer immunotherapy.

Emerging data from a wide range of studies on acute and chronic infections support an important role for negative costimulatory receptors also in controlling infection. Memory CD8 T cells generated after an acute viral infection are highly functional and constitute an important component of protective immunity. Modulation of costimulatory pathway has also been proven effective in optimizing antiviral immunity by limiting the memory T cell response to its protective capacities (Teijaro et al., J Immunol. 2009: 182; 5430-5438). This has been demonstrated in models of influenza infection in which inhibiting CD28 costimulation with CTLA4-Ig suppressed primary immune responses in naive mice infected with influenza, but was remarkably curative for memory CD4 T cell-mediated secondary responses to influenza leading to improved clinical outcome and increased survival to influenza challenge.

Chronic infections are often characterized by varying degrees of functional impairment of virus-specific T-cell responses, and this defect is a principal reason for the inability of the host to eliminate the persisting pathogen. Although functional effector T cells are initially generated during the early stages of infection, they gradually lose function during the course of the chronic infection as a result of persistant exposure to foreign antigen, giving rise to T cell exhaustion. Exhausted T cells express high levels of multiple co-inhibitory receptors such as CTLA-4, PD-1, and LAG3 (Crawford et al., Curr Opin Immunol. 2009;21 : 179-186; Kaufmann et al., J Immunol 2009;182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). PD-1 overexpression by exhausted T cells was observed clinically in patients suffering from chronic viral infections including HIV, HCV and HBV (Crawford et al., Curr Opin Immunol 2009;21: 179-186; Kaufmann et al.,

J Immunol 2009;182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). There has been some investigation into this pathway in additional pathogens, including other viruses, bacteria, and parasites (Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694, Bhadra et al., Proc Natl Acad Sci. 2011 ;108(22):9196-201). For example, the PD-1 pathway was shown to be involved in controlling bacterial infection using a sepsis model induced by the standard cecal ligation and puncture method. The absence of PD-1 in knockout mice protected from sepsis-induced death in this model (Huang et al., PNAS 2009: 106; 6303-6308).

T cell exhaustion can be reversed by blocking co-inhibitory pathways such as PD-1 or CTLA-4 (Rivas et al., J Immunol. 2009 ;183:4284-91 ; Golden-Mason et al., J Virol. 2009;83:9122-30; Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694), thus allowing restoration of anti viral immune function. The therapeutic potential of co-inhibition blockade for treating viral infection was extensively studied by blocking the PD-l/PD-Ll pathway, which was shown to be efficacious in several animal models of infection including acute and chronic simian immunodeficiency virus (SIV) infection in rhesus macaques (Valu et al., Nature 2009;458:206-210) and in mouse models of chronic viral infection, such as lymphocytic choriomeningitis virus (LCMV) (Barber et al., Nature. 2006;439:682-7), and Theiler's murine encephalomyelitis virus (TMEV) model in SJL/J mice (Duncan and Miller PLoS One. 201 l ;6:el 8548). In these models PD-l/PD-LI blockade improved anti viral responses and promoted clearance of the persisting viruses. In addition, PD-l/PD-Ll blockade increased the humoral immunity manifested as elevated production of specific anti-virus antibodies in the plasma, which in combination with the improved cellular responses leads to decrease in plasma viral loads and increased survival.

Blocking negative signaling pathways, such as PD-1 and CTLA-4, can restore the host immune system, enabling it to respond to further stimulation. Combining therapeutic vaccination along with the blockade of inhibitory signals could synergistically enhance functional CD8 T-cell responses and improve viral control in chronically infected individuals, providing a promising strategy for the treatment of chronic viral infections, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus (Ha et al, Immunol Rev. 2008 Jun; 223:317-33). The results of a recent study indicate that blocking of the PD-1 pathway improved T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this pathway might improve

success rates of immunization in the setting of chronic viral infection (Moorman et al, Vaccine. 2011 Apr 12;29(17):3169-76). Antibodies to PD-1 and CTLA-4 are currently in clinical trials in chronic hepatitis C, as promising candidates for combination with both prophylactic and therapeutic vaccines (Diepolder and Obst, Expert Rev Vaccines. 2010 Mar;9(3):243-7). PD-1 blockade also enhances the effectiveness of prophylactic vaccination leading to an increase in epitope specific T cells (Finnefrock et al., J Immunol 2009;182;980-987)

In addition to blockade of co-inhibitory pathways for treatment of chronic infections, recent studies using viral infection models have highlighted the importance of positive costimulatory signals during memory responses against viruses. Costimulatory molecules such as CD28, 4-1BB, and OX40 have also been implicated in the survival, generation, maintenance, and quality of virus-specific memory CD8+T cells. The delivery of costimulatory signals can help boost the generation and function of virus-specific memory CD8+ T cells. The use of costimulatory molecules as adjuvants, along with viral antigens in vaccines, may facilitate the generation of effective antigen-specific memory CD8+ T-cell responses, and may therefore lead to improved vaccines (Duttagupta et al, Crit Rev Immunol. 2009;29(6):469-86).

A recent study also evaluated the effects of soluble PD-1 (sPD-1) as a blockade of PD-1 and PD-L1 on vaccine-elicited antigen-specific T-cell responses in mice. Coadministration of sPD-1 with a DNA vaccine or with an adeno virus-based vaccine, increased antigen-specific CD8(+) T-cell responses, indicating vaccine type-independent adjuvant effect of sPD-1 (Song et al, J Immunother. 2011 Apr;34(3):297-306). These and additional results of this study suggest that an immunization strategy using the soluble extracellular domain (ECD) of a negative costimulatory protien as an adjuvant, could be used to increase antigen-specific T-cell immunity elicited by vaccination.

B cells have also long been considered to have a key role in the development and maintenance of many autoimmune diseases through production of pathogenic autoantibodies, such as systemic lupus erythematosus (SLE) and Sjogren's disease. However, it is clear that a number of other B cell functions are also critical in the pathogenesis of organ-specific autoimmune diseases that were previously thought to be mainly T cell mediated, such as rheumatoid arthritis (RA) and type 1 diabetes (T1D) (Wong et al 2010, Curr Opin Immunol. 22:723-731).

T cell help to B cells is a pivotal process of adaptive immune responses. Follicular helper T (Tfh) cells are a subset of CD4+ T cells specialized in B cell help (reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663, 2011). Tfh cells express the B cell homing chemokine receptor, CXCR5, which drives Tfh cell migration into B cell follicles within lymph nodes in a CXCL 13 -dependent manner. Tfh cells first interact with cognate B cells at the T cell-B cell border and subsequently induce germinal center B cell differentiation and germinal center formation within the follicle (Reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663, 2011). The requirement of Tfh cells for B cell help and T cell-dependent antibody responses indicates that this cell type is of great importance for protective immunity against various types of infectious agents, as well as for rational vaccine design. Not surprisingly, dysregulation and aberrant accumulation of Tfh cells has also been linked with autoimmune diseases, such as Sjogren's disease and autoimmune arthritis (Yu and Vinuesa, 2010, Cell. Mol. Immunol. 7: 198-203).

Tfh cells selectively express a wealth of surface proteins, which are involved in their selective localization (such as CXCR5) and in direct physical interactions with B cells to provide B cell help. Among the latter group are several members of the costimulatory proteins family which are highly expressed in Tfh cells, including the inducible co-stimulatory receptor ICOS, and the negative costimulators (inhibitory receptors) PD-1 and BTLA (Crotty, Annu. Rev. Immunol. 29: 621-663, 2011), thus this cell subset may be also controlled by modulation of costimulatory and coinhibitory pathways, contributing to the effect on B cell function.

Regulating costimulation using agonists and/or antagonists to various costimulatory proteins has been extensively studied as a strategy for treating autoimmune diseases, graft rejection, allergy and cancer. This field has been clinically pioneered by CTLA4-Ig (Abatacept, Orencia®) which is approved for treatment of RA, mutated CTLA4-Ig (Belatacept, Nulojix®) for prevention of acute kidney transplant rejection and by the anti-CTLA4 antibody (Ipilimumab, Yervoy®), recently approved for the treatment of melanoma. Other costimulation regulators are currently in advanced stages of clinical development including anti-PD-1 antibody (MDX-1106) which is in development for treatment of advanced/metastatic clear-cell renal cell carcinoma (RCC) and anti-CD40L Antibody (BG9588, Antova®) for treatment of renal allograft transplantation. Furthermore, such agents are also in clinical development for viral infections, for example the anti PD-1 Ab, MDX-1106, which is being tested for treatment of hepatitis C, and the anti-CTLA-4 Ab CP-675,206 (tremelimumab) which is in a clinical trial in hepatitis C virus-infected patients with hepatocellular carcinoma; the goals of the study are to test its effect on the carcinoma and on the replication of the virus.

BRIEF SUMMARY OF THE INVENTION

According to at least some embodiments, the invention provides novel therapeutic and diagnostic compositions containing an ectodomain or soluble or secreted form of the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins and/or variants and/or orthologs and/or fragments, and/or conjugate containing same, and/or nucleic acid sequences encoding for same.

The full length amino acid sequence of the known (wild type) LY6G6F protein (lymphocyte antigen 6 complex locus protein G6f, genbank accession number: NP_001003693, SEQ ID NO: l) is shown in Figure 1A. The full length amino acid sequence of known (wild type) VSIGIO protein (V-set and immunoglobulin domain-containing protein 10, genbank accession number: NP_061959, SEQ ID NO:3), and the amino acid sequence of VSIGIO novel variant (SEQ ID NO:5) are shown in Figures IB and 1C, respectively. The amino acid sequence alignment of VSIGIO novel variant (SEQ ID NO: 5) and the known (wild type) VSIGIO protein (SEQ ID NO: 3) is shown in Figure 2A. The full length amino acid sequence of known (wild type) TMEM25 protein (Transmembrane protein 25, Swiss-Prot accession number: Q86YD3, SEQ ID NO:7) is shown in Figure ID. The full length amino acid sequence of known (wild type) LSR protein (lipolysis-stimulated lipoprotein receptor isoform 2, genbank accession number: NP_991403) is provided in SEQ ID NO:62. The amino acid sequences of LSR variants SEQ ID NOs:l l, 13, 15, 16, 17 and 18 are shown in Figures IE, IF, 1G, 1H, II, and 1J, respectively. The amino acid sequence alignment of the LSR variants SEQ ID NOs: 11, 13, 15, 16, 17 and 18 with previously known LSR sequences (SEQ ID NOs: 62-67) is demonstrated in Figures 2B, 2C, 2D, 2E, 2F, 2G, respectively.

According to at least some embodiments, there is provided an isolated polypeptide comprising at least 98 amino acids of the soluble ectodomain of a sequence selected from the group consisting of SEQ ID NOs: l l, 13, 15-18, 67, and 143; at least 62 amino acids of the soluble ectodomain of a sequence selected from the group consisting of SEQ ID NOs: l and 58; at least 36 amino acids of the soluble ectodomain of a sequence selected from the group consisting of SEQ ID NOs: 3 and 5; or at least 46

amino acids of the soluble ectodomain of SEQ ID NO:7, or an isolated polypeptide consisting essentially of an amino acid sequence as set forth in SEQ ID NO: 5 or variant thereof that possesses at least 95% sequence identity therewith; or variants, or orthologs, or fragments thereof.

Optionally the isolated polypeptide comprises only between 98 to 180 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l l, 13, 15-18, 67, and 143; between 62 to 228 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l and 58; between 36 and 393 of the sequence selected from the group consisting of SEQ ID NOs:3 and 5; or between 46 and 216 amino acids of SEQ ID NO:7.

Also optionally, the isolated polypeptide is selected from the group consisting of a polypeptide comprising only between 98 to 118, 135 to 155, and 160 to 180 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l l, 13, 15-18, 67, and 143; between 62 to 82, 95 to 115, 208 to 228 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l and 58; between 36 to 70, 80 to 100, 170 to 200, 265 to 290, 365 to 393 amino acids of the sequence selected from the group consisting of SEQ ID NOs:3 and 5; or between 46 to 66, 84 to 104, 196 to 216 amino acids of SEQ ID NO:7.

Also optionally, the isolated polypeptide comprises only about 72, 106, or 218 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l and 58; about 108, 145, or 170 amino acids of the sequence selected from the group consisting of SEQ ID NOs: l l, 13, 15-18, 67, and 143; about 56, 94, or 206 amino acids of SEQ ID NO:7; or about 46,49,58,60, 87, 89, 93, 94, 178, 182, 185, 187, 273, 279, 282, 374 or 383 amino acids of SEQ ID NOs:3 and 5.

Also optionally, the isolated polypeptide consists essentially of an amino acid sequence having at least 95% sequence identity with amino acid sequences set forth in any one of SEQ ID NOs: 12, 2, 4-6, 8, 14, 47-50, 10, 15-18, 22, 39, 59-61; 81-102. Optionally and preferably, the isolated polypeptide consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 12, 2, 4-6, 8, 14, 47-50, 10, 15-18, 22, 39, 59-61 ; 81-102.

Optionally, the isolated polypeptide blocks or inhibits the interaction of LSR, TMEM25, VSIG10, LY6G6F, or a fragment or variant thereof with a corresponding functional counterpart.

Optionally, the isolated polypeptide replaces or augments the interaction of LSR, TMEM25, VSIGIO, LY6G6F, or a fragment or variant thereof with a corresponding functional counterpart.

Optionally, the isolated ortholog is a mouse polypeptide selected from SEQ ID NOs: 9 and 19-21.

According to at least some embodiments, the present invention provides isolated polypeptides comprising discrete portions (fragments) of VSIGIO proteins, corresponding to:

A. An isolated chimeric polypeptide, comprising a first amino acid sequence being at least 95% homologous to

MAAGGSAPEPRVLVCLGALLAGWVAVGLEAVVIGEVHENVTLHCGNISGLRGQ VTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGIYTCQEILNVT QWFQVWLQVA corresponding to amino acids 1 - 120 of known VSIGIO protein (SEQ ID NO:3), which also corresponds to amino acids 1 - 120 of VSIGIO variant (SEQ ID NO:5), a second bridging amino acid sequence comprising of N, and a third amino acid sequence being at least 95% homologous to

PPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVE

MLSESQLSDGKKFKCVTSHIVGPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLT

CQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYI CRADSPVGVREMEIWLSVKEPLNIGGIVGTIVSLLLLGLAIISGLLLHYSPVFCWK VGNTSRGQNMDDVMVLVDSEEEEEEEEEEEEDAAVGEQEGAREREELPKEIPKQ DHIHRVTALVNGNIEQMGNGFQDLQDDSSEEQSDIVQEEDRPV corresponding to amino acids 223 - 540 of known VSIGIO protein (SEQ ID NO:3), which also corresponds to amino acids 122 - 439 of VSIGIO variant (SEQ ID NO:5), wherein said first amino acid sequence, second bridging amino acid sequence and third amino acid sequence are contiguous and in a sequential order.

B. An isolated polypeptide of an edge portion of VSIGIO variant (SEQ ID NO:5), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least 3 amino acids comprise ANP having a structure as follows (numbering according to VSIGIO variant

(SEQ ID NO:5)): a sequence starting from any of amino acid numbers 120-x to 120; and ending at any of amino acid numbers 122 + ((n-3) - x), in which x varies from 0 to n-3.

According to at least some embodiments, the subject invention further provides isolated polypeptides comprising a sequence of amino acid residues corresponding to discrete portions of VSIGIO proteins, corresponding to the new junction and edge portions of VSIGIO variant (SEQ ID NO: 5). The unique sequence of the new junction of VSIGIO variant (SEQ ID NO: 5) is demonstrated in protein sequence alignment in Figure 2A.

According to at least some embodiments, the subject invention provides isolated polypeptides comprising discrete portions (fragments) of LSR proteins, corresponding to:

A. An isolated chimeric polypeptide, comprising a first amino acid sequence being at least 95% homologous to

MALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVV ILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIAD AFSPASVDNQLN AQLAAGN PGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAW GDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPGFQAGPIE

corresponding to amino acids 49 - 258 of known LSR protein (SEQ ID NO: 62), which also corresponds to amino acids 1 - 210 of LSR variant isoform f (SEQ ID NO: 18), a second bridging amino acid sequence comprising of V, and a third amino acid sequence being at least 95% homologous to

YAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRS SSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDP SRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWD QEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPP RSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRS GDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRL KKNLALSRESLVV corresponding to amino acids 309 - 649 of known LSR protein (SEQ ID NO:62), which also corresponds to amino acids 212 - 552 of LSR variant isoform f (SEQ ID NO: 18), wherein said first amino acid sequence, second bridging amino acid and third amino acid sequence are contiguous and in a sequential order.

B. An isolated polypeptide of an edge portion of LSR variant isoform f (SEQ ID NO:18), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least 3 amino acids comprise EVY having a structure as follows (numbering according to SEQ ID NO: 18): a sequence starting from any of amino acid numbers 210-x to 210; and ending at any of amino acid numbers 212 + ((n-3) - x), in which x varies from 0 to n-3.

C. An isolated chimeric polypeptide comprising a first amino acid sequence being at least 95% homologous to

MALLAGGLSRGLGSHPAAAGRDAVVFVWLLLSTWCTAPARAIQVTVSNPYHVV ILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGN PGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAW GDSGVYYCSVVSAQDLQGNNEAYAELIVL corresponding to amino acids 49 - 239 of known LSR protein (SEQ ID NO:66), which also corresponds to amino acids 1 - 191 of LSR variant isoform f (SEQ ID NO: 18), a second amino acid sequence being at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GRTSGVAELLPGFQAGPIE corresponding to amino acids 192 - 218 of LSR variant isoform f (SEQ ID NO: 18), and a third amino acid sequence being at least 95% homologous to

VYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVD RSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANF DPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRG WDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAY MPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDN GSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASR ERRLKKNLALSRESLVV corresponding to amino acids 240 - 581 of known LSR protein SEQ ID NO:66, which also corresponds to amino acids 211 - 552 of LSR variant isoform f (SEQ ID NO: 18), wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.

D. An isolated polypeptide of an edge portion of LSR variant isoform f (SEQ ID NO: 18), comprising an amino acid sequence being at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GRTSGVAELLPGFQAGPIE of LSR variant isoform f (SEQ ID NO: 18).

According to at least some embodiments, the subject invention further provides isolated polypeptides comprising a sequence of amino acid residues corresponding to discrete portions of LSR, corresponding to the new junction and edge portions of LSR variant LSR isoform-f (SEQ ID NO: 18). The unique sequences of the new junction of the LSR isoform-f (SEQ ID NO: 18) is demonstrated in protein sequence alignment in Figure 2G.

According to at least some embodiments, the subject invention provides polypeptides comprising a sequence of amino acid residues corresponding to discrete portions of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins, including different portions of the extracellular domain corresponding to residues 17-234 of LY6G6F (SEQ ID NO: l), corresponding to amino acid sequence depicted in SEQ ID NO:2; residues 31-413 of VSIGIO (SEQ ID NO:3), corresponding to amino acid sequence depicted in SEQ ID NO:4; residues 31-312 of VSIGIO (SEQ ID NO:5), corresponding to amino acid sequence depicted in SEQ ID NO:6; residues 27-232 of TMEM25 (SEQ ID NO:7), corresponding to amino acid sequence depicted in SEQ ID NO:8; residues 42-211 of LSR (SEQ ID NO: 11, and/or SEQ ID NO: 143), corresponding to amino acid sequence depicted in SEQ ID NO: 12; residues 42-192 of LSR (SEQ ID NO: 13), corresponding to amino acid sequence depicted in SEQ ID NO: 14, residues 42-533 of LSR (SEQ ID NO:15), corresponding to amino acid sequence depicted in SEQ ID NO:47, residues 42-532 of LSR (SEQ ID NO: 16), corresponding to amino acid sequence depicted in SEQ ID NO:48, residues 42-493 of LSR (SEQ ID NO: 17), corresponding to amino acid sequence depicted in SEQ ID NO:49, residues 42-552 of LSR (SEQ ID NO: 18), corresponding to amino acid sequence depicted in SEQ ID NO: 50, and/or fragments and/or variants thereof possessing at least 85%, 90%, 95, 96, 97, 98 or 99% sequence homology therewith. According to still further embodiments, the LY6G6F ECD fragments are selected from any one of SEQ ID NOs 81, 96, and variants thereof, as described herein. According to still further embodiments, the VSIGIO ECD fragments are selected from any one of SEQ ID NOs 82-93, 97-100, and variants thereof, as described herein. According to still further embodiments, the LSR ECD fragments are selected from any one of SEQ ID NOs 95, 102, and variants thereof, as described herein. According to still further embodiments, the TMEM25 ECD fragments are selected from any one of SEQ ID NOs 94, 101, and

variants thereof, as described herein. According to still further embodiments, the discrete portions of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins may or may not include a signal (leader) peptide (SP) sequence (Figure 1). According to at least some embodiments of the invention, there are provided examples of the ECD portions including SP sequences of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins. An example of ECD portion including SP sequence of LY6G6F protein (SEQ ID NO: l) is amino acid sequence set forth in SEQ ID NO:59. An example of ECD portion including SP sequence of VSIGIO protein (SEQ ID NO: 3) is amino acid sequence set forth in SEQ ID NO: 60. An example of ECD portion including SP sequence of VSIGIO protein (SEQ ID NO:5) is amino acid sequence set forth in SEQ ID NO:61. An example of ECD portion including SP sequence of TMEM25 protein (SEQ ID NO:7) is amino acid sequence set forth in SEQ ID NO: 39. An example of ECD portion including SP sequence of LSR protein (SEQ ID NO: 11) is amino acid sequence set forth in SEQ ID NO: 10. An example of ECD portion including SP sequence of LSR protein (SEQ ID NO: 14) is amino acid sequence set forth in SEQ ID NO:22.

According to further embodiments, the invention provides polypeptides comprising a sequence of amino acid residues corresponding to soluble LSR proteins depicted in SEQ ID NO: 18, including different portions thereof or variants thereof possessing at least 85%, 90%, 95, 96, 97, 98 or 99% sequence homology therewith. According to further embodiments, the invention provides polypeptides comprising a sequence of amino acid residues corresponding to soluble LSR proteins depicted in any one of SEQ ID NOs: 15-16, including different portions thereof or variants thereof possessing at least 95, 96, 97, 98 or 99% sequence homology therewith. According to further embodiments, the invention provides polypeptides comprising a sequence of amino acid residues corresponding to soluble LSR proteins depicted in any one of SEQ ID NOs: 15-18. According to still further embodiments, the soluble LSR proteins depicted in any one of SEQ ID NOs: 15-18 may or may not include a signal (leader) peptide sequence (Figure 1G, G, I and J).

According to still further embodiments, the invention provides polypeptides comprising a sequence of amino acid residues corresponding to extracellular domains of orthologs of TMEM25, LY6G6F, VSIGIO, LSR variant 1 and/or LSR variant 2 proteins, particularly mouse orthologs (SEQ ID NOs: 28, 29, 30, 31 and/or 32, respectively), including but not limited to mouse orthologs extracellular domains corresponding to

amino acid sequence depicted in SEQ ID NOs: 9, 19-21, or portions or variants thereof possessing at least 85%, 90%, 95, 96, 97, 98 or 99% sequence homology therewith.

According to still further embodiments, the invention provides polypeptides comprising an amino acid sequence corresponding to any one of novel variants of VSIG10 (SEQ ID NO: 5), and LSR (SEQ ID NOs: 11, 13, 15, 16 and 18).

According to at least some embodiments, the present invention provides a fusion protein comprising any of the above polypeptides joined to a heterologous sequence. Optionally, the heterologous sequence comprises at least a portion of an immunoglobulin molecule. Optionally and preferably, the immunoglobulin molecule portion is an immunoglobulin heavy chain constant region Fc fragment. Optionally and more preferably, the immunoglobulin heavy chain constant region is derived from an immunoglobulin isotype selected from the group consisting of an IgGl, IgG2, IgG3, IgG4, IgM, IgE, IgA and IgD. Optionally and most preferably, the fusion protein has the amino acid sequence set forth in any one of SEQ ID NOs: 71-80, 172-181 or set forth in any one of SEQ ID NOs:23-26 and also optionally modulates immune cell response in vitro or in vivo.

According to at least some embodiments, the subject invention provides isolated nucleic acid sequences encoding any one of the foregoing novel variants of TMEM25, VSIG10, and/or LSR and/or any one of the foregoing LY6G6F, VSIG10, TMEM25 and/or LSR extracellular domain polypeptides or fragments or homologs or orthologs thereof.

According to at least some embodiments, there is provided an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33-37, 40-46, 132, 155, 182-198, or variant thereof that possesses at least 95% sequence identity therewith, or a degenerative variant thereof.

According to at least some embodiments, the subject invention provides an isolated polynucleotide encoding a polypeptide comprising any one of the amino acid sequences, as set forth in SEQ ID NOs: 2, 4, 5, 6, 8-16, 18-22, 39, 47-50, 59-61, 143, or a fragment or variant thereof that possesses at least 85, 90, 95, 96, 97, 98 or 99% sequence identity therewith, or a degenerative variant thereof.

According to at least some embodiments, the subject invention provides an isolated polynucleotide comprising a nucleic acid as set forth in any one of SEQ ID NO:33-37, 40-46, 132, 145, 155, 182-188, or a sequence homologous thereto or

degenerative variants thereof. According to another embodiment, the isolated polynucleotide is at least 85, 90, 95, 96, 97, 98 or 99% homologous to a nucleic acid sequence as set forth in any one of SEQ ID NOs: 33-37, 40-46, 145.

According to at least some embodiments, there is provided an expression vector or a virus, containing at least one isolated nucleic acid sequence as described herein. According to at least some embodiments, there is provided a recombinant cell comprising an expression vector or a virus containing an isolated nucleic acid sequence as described herein, wherein the cell constitutively or inducibly expresses the polypeptide encoded by the DNA segment. According to at least some embodiments, there is provided a method of producing a LSR, TMEM25, VSIG10, LY6G6F soluble ectodomain polypeptide, or fragment or fusion protein thereof, comprising culturing the recombinant cell as described herein, under conditions whereby the cell expresses the polypeptide encoded by the DNA segment or nucleic acid and recovering said polypeptide.

According to at least some embodiments of the present invention, there is provided a pharmaceutical composition comprising an isolated amino acid sequence of ectodomain or soluble or secreted forms of any one of LY6G6F, VSIG10, TMEM25, LSR proteins or variants or orthologs or fragments or conjugates containing same.

According to at least some embodiments, the invention provides an isolated or purified amino acid sequence of soluble and/or extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR protein or nucleic acid sequence encoding same, which optionally may be directly or indirectly attached to a non-LY6G6F, VSIG10, TMEM25 and/or LSR protein or nucleic acid sequence, such as a soluble immunoglobulin domain or fragment.

According to at least some embodiments, the invention provides vectors such as plasmids and recombinant viral vectors and host cells containing that express secreted or soluble form and/or the ECD of the LY6G6F, VSIG10, TMEM25 and/or LSR protein or fragments or variants or orthologs thereof or polypeptide conjugates containing any of the foregoing.

According to at least some embodiments the invention provides a use of these vectors such as plasmids and recombinant viral vectors and host cells containing that express any one of LY6G6F, VSIG10, TMEM25 and/or LSR, secreted and/or soluble form and/or the ECD and/or fragments thereof and/or variants, and/or orthologs thereof and/or polypeptide conjugates containing any of the foregoing to produce any one of said LY6G6F, VSIGIO, TMEM25 and/or LSR proteins.

According to at least some embodiments, the invention provides pharmaceutical or diagnostic compositions containing any of the foregoing.

According to at least some embodiments, the invention provides a use of any one of the compounds containing at least one of LY6G6F, VSIGIO, TMEM25 and/or LSR ectodomains, soluble or secreted form or fragments or orthologs or variants thereof, or conjugates, or nucleic acid sequence encoding same, or pharmaceutical composition comprising same, as therapeutics for treatment or prevention of cancer as recited herein, infectious disorder as recited herein, and/or immune related disorder, including but not limited to autoimmune diseases as recited herein, transplant rejection and graft versus host disease and/or for blocking or promoting immune costimulation mediated by any one of the LY6G6F, VSIGIO, TMEM25 and/or LSR polypeptides, immune related diseases as recited herein and/or for immunotherapy (promoting or inhibiting immune costimulation). According to at least some embodiments, the autoimmune disease includes any autoimmune disease, and optionally and preferably includes but is not limited to any of the types and subtypes of any of multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, or Sjogren's syndrome.

According to at least some embodiments, the invention provides a use of any one of the compounds containing at least one of LY6G6F, VSIGIO, TMEM25 and/or LSR ectodomains, soluble or secreted form or fragments or orthologs or variants thereof, or conjugates, or nucleic acid sequence encoding same, or pharmaceutical composition comprising same, for administration as an anti-cancer vaccine, as an adjuvant for anti cancer vaccine, and/or for adoptive immunotherapy, and/or for immunotherapy of cancer as recited herein.

According to at least some embodiments, the invention provides a use of any of the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins, and/or nucleic acid sequences as targets for development of drugs which specifically bind to any one of the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins and/or drugs which agonize or antagonize the binding of other moieties to the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins.

According to at least some embodiments, the present invention provides drugs which modulate (agonize or antagonize) at least one of the LY6G6F, VSIGIO, TMEM25

and/or LSR related biological activity. Such drugs include by way of example antibodies, small molecules, peptides, ribozymes, aptamers, antisense molecules, siRNA's and the like. These molecules may directly bind or modulate an activity elicited by the any one of the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins or the LY6G6F, VSIGIO, TMEM25 and/or LSR DNA or portions or variants thereof or may indirectly modulate any one of the LY6G6F, VSIGIO, TMEM25 and/or LSR associated activity or binding of molecules to any one of the LY6G6F, VSIGIO, TMEM25 and/or LSR and portions and variants thereof such as by modulating the binding of any one of LY6G6F, VSIGIO, TMEM25 and/or LSR to its counterreceptor or endogenous ligand.

According to at least some embodiments, the invention provides novel monoclonal or polyclonal antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, that specifically bind any one of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins as described herein or polypeptides having at least 95% homology thereto. Optionally such antibodies bind to proteins selected from the group consisting of any one of SEQ ID NOs: 1-8, 10-18, 22, 39, 47-50, 59-61, 9, 19-21, and/or the amino acid sequences corresponding to the unique edges of any one of SEQ ID NOs: 5 and 18, particularly wherein these antibodies, antigen binding fragments and conjugates containing same, and/or alternative scaffolds, are adapted to be used as therapeutic and/or diagnostic agents (both in vitro and in vivo diagnostic methods), particularly for treatment and/or diagnosis of infectious disorder as recited herein, and/or immune related disorder, including but not limited to autoimmune diseases as recited herein, immune related diseases as recited herein, transplant rejection and graft versus host disease, as well as cancers and malignancies as recited herein.

According to at least some embodiments, there are provided antibodies in which the antigen binding site comprises a conformational or linear epitope, and wherein the antigen binding site contains about 3-7 contiguous or non-contiguous amino acids. Optionally, the antibody is a fully human antibody, chimeric antibody, humanized or primatized antibody.

Also optionally, the antibody is selected from the group consisting of Fab, Fab', F(ab')2, F(ab'), F(ab), Fv or scFv fragment and minimal recognition unit.

Also optionally, the antibody is coupled to a moiety selected from a drug, a radionuclide, a fluorophore, an enzyme, a toxin, a therapeutic agent, or a chemotherapeutic agent; and wherein the detectable marker is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound.

Also optionally the antibody blocks or inhibits the interaction of any one of LSR, TMEM25, VSIGIO, LY6G6F polypeptides, or a fragment or variant thereof with a counterpart.

Also optionally the antibody replaces or augments the interaction of LSR, TMEM25, VSIGIO, LY6G6F polypeptides, or a fragment or variant thereof with a counterpart.

Also optionally the antibody elicits apoptosis or lysis of cancer cells that express any one of LSR, TMEM25 , VSIG 10, LY6G6F protein.

Also optionally the apoptosis or lysis involves CDC or ADCC activity of the antibody, wherein CDC (complement dependent cytotoxicity) or ADCC (antibody dependent cellular cytotoxicity) activities are used to target the immune cells.

According to at least some embodiments, the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the LY6G6F protein including different portions of the extracellular domain corresponding to residues 17-234 of LY6G6F (SEQ ID NO: l), set forth in SEQ ID NO: 2, and/or corresponding to amino acid sequences set forth in any one of SEQ ID NOs: 81, 96. According to further embodiments the invention provides antibodies antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the mouse LY6G6F protein (SEQ ID NO: 29), including different portions of the extracellular domain corresponding to SEQ ID NO:20.

According to at least some embodiments, the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the VSIGIO protein including different portions of the extracellular domain corresponding to amino acid residues 31-413 of VSIGIO (SEQ ID NO:3), depicted in SEQ ID NO:4; amino acid residues 31-312 of VSIGIO (SEQ ID NO:5), depicted in SEQ ID NO:6, and/or corresponding to amino acid sequences set forth in any one of SEQ ID NOs:82-93, 97-100. According to further embodiments the invention provides antibodies antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the mouse VSIGIO protein (SEQ ID NO: 30), including different portions of the extracellular domain corresponding to SEQ ID NO: 19. According to at least some embodiments, the invention provides

antibodies, antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the VSIGIO protein including the edge portion of VSIGIO variant (SEQ ID NO:5), as described herein.

According to at least some embodiments, the invention provides antibodies, antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the TMEM25 proteins including different portions of the extracellular domain corresponding to amino acid residues 27-232 of TMEM25 (SEQ ID NO:7), depicted in SEQ ID NO:8, and/or corresponding to amino acid sequences set forth in any one of SEQ ID NOs: 94, 101. According to further embodiments the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the mouse TMEM25 protein (SEQ ID NO: 28), including different portions of the extracellular domain, set forth in SEQ ID NO:9.

According to at least some embodiments, the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the LSR proteins including different portions of the extracellular domain corresponding to amino acid residues 42-211 of LSR (SEQ ID NO:l l), depicted in SEQ ID NO: 12; amino acid residues 42-192 of LSR (SEQ ID NO:13), depicted in SEQ ID NO: 14, amino acid residues 42-533 of LSR (SEQ ID NO:15), depicted in SEQ ID NO:47, amino acid residues 42-532 of LSR (SEQ ID NO:16), depicted in SEQ ID NO:48, amino acid residues 42-493 of LSR (SEQ ID NO: 17), depicted in SEQ ID NO:49, amino acid residues 42-552 of LSR (SEQ ID NO: 18), depicted in SEQ ID NO:50, and/or corresponding to amino acid sequences set forth in any one of SEQ ID NOs:95, 102. According to further embodiments the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the mouse LY6G6F proteins (SEQ ID NOs: 31-32), including different portions of the extracellular domain corresponding to SEQ ID NO:21.

According to at least some embodiments, the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the LSR proteins including the unique edge portion of LSR variant isoform-f (SEQ ID NO: 18), as described herein.

According to at least some embodiments, the invention provides antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against discrete portions of the soluble LSR proteins including different portions of the LSR proteins depicted in any one of SEQ ID NOs: 15-18, 47-50.

According to at least some embodiments the invention relates to protein scaffolds with specificities and affinities in a range similar to specific antibodies. According to at least some embodiments the present invention relates to an antigen-binding construct comprising a protein scaffold which is linked to one or more epitope-binding domains. Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques. According to at least some embodiments the invention relates to alternative scaffolds including, but not limited to, anticalins, DARPins, Armadillo repeat proteins, protein A, lipocalins, fibronectin domain, ankyrin consensus repeat domain, thioredoxin, chemically constrained peptides and the like. According to at least some embodiments the invention relates to alternative scaffolds that are used as therapeutic agents for treatment of cancer as recited herein, immune related diseases as recited herein, autoimmune disease as recited herein and infectious diseases, as well as for in vivo diagnostics.

According to at least some embodiments of the present invention, there is provided a pharmaceutical composition comprising an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein, and further comprising a pharmaceutically acceptable diluent or carrier.

According to at least some embodiments, there is provided use of any of any one of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition as described herein, wherein administration of such to the subject inhibits or reduces activation of T cells.

According to at least some embodiments, there is provided use of any of any one of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition as described herein, for treatment of cancer.

According to at least some embodiments, there is provided use of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition as described herein, for treatment of infectious disorder.

According to at least some embodiments, there is provided a method of performing one or more of the following in a subject:

a. upregulating cytokines;

b. inducing expansion of T cells;

c. promoting antigenic specific T cell immunity;

d. promoting CD4+ and/or CD8+ T cell activation;

comprising administering any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition as described hereinto the subject.

According to at least some embodiments, there is provided a method for treating or preventing immune system related condition comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition.

Optionally, the immune system related condition comprises an immune related condition, autoimmune diseases as recited herein, transplant rejection and graft versus host disease and/or for blocking or promoting immune costimulation mediated by any one of the LSR, TMEM25, VSIG10, and/or LY6G6F polypeptides, immune related diseases as recited herein and/or for immunotherapy (promoting or inhibiting immune costimulation).

Optionally the treatment is combined with another moiety useful for treating immune related condition.

Optionally the moiety is selected from the group consisting of immunosuppressants such as corticosteroids, cyclosporin, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, biological agents such as TNF-alpha blockers or antagonists, or any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, Cytoxan, interferon beta-la, interferon beta-lb, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologies and/or intravenous immunoglobulin (IVIG), interferons such as IFN-beta-la (REBIF®. and AVONEX®) and IFN-beta-lb (BETASERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONE®), a cytotoxic agent, a calcineurin inhibitor, e.g. cyclosporin A or FK506; an immunosuppressive macrolide, e.g. rapamycine or a derivative thereof; e.g. 40-O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720 or an analog thereof, corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD l la/CD18, CD7, CD25, CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig (abatacept, ORENCIA®), CD28-Ig, B7-H4-Ig, or other costimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists, or another immunomodulatory agent.

Optionally the immune condition is selected from autoimmune disease, transplant rejection, or graft versus host disease.

Optionally the autoimmune disease is selected from a group consisting of multiple sclerosis, including relapsing-remiting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis, systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic angiitis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red cell aplasia, Sjogren's syndrome, rheumatic disease, connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, juvenile rheumatoid arthritis, arthritis uratica, muscular rheumatism, chronic polyarthritis, cryoglobulinemic vasculitis, ANCA-associated vasculitis, antiphospholipid syndrome, myasthenia gravis, autoimmune haemolytic anaemia, Guillian-Barre syndrome, chronic immune polyneuropathy, autoimmune thyroiditis, insulin dependent diabetes mellitus, type I diabetes, Addison's disease, membranous glomerulonephropathy, Goodpasture's disease, autoimmune gastritis, autoimmune atrophic gastritis, pernicious anaemia, pemphigus, pemphigus vulgarus, cirrhosis, primary biliary cirrhosis, dermatomyositis, polymyositis, fibromyositis, myogelosis, celiac disease, immunoglobulin A nephropathy, Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis, psoriasis, psoriasis arthropathica, Graves' disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma, progressive systemic scleroderma, asthma, allergy, primary biliary cirrhosis, Hashimoto's thyroiditis, primary myxedema, sympathetic ophthalmia, autoimmune uveitis, hepatitis, chronic action hepatitis, collagen diseases, ankylosing spondylitis, periarthritis humeroscapularis, panarteritis nodosa, chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis, chronic urticaria, bullous skin disorders, pemphigoid, atopic eczema, Devic's disease, childhood autoimmune hemolytic anemia, Refractory or chronic Autoimmune Cytopenias, Prevention of development of Autoimmune Anti-Factor VIII Antibodies in Acquired Hemophilia A, Cold Agglutinin Disease, Neuromyelitis Optica, Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne, normocomplementemic urticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler's syndrome, autoimmune retinopathy, age-related macular degeneration, atherosclerosis, chronic prostatitis and TNF receptor-associated periodic syndrome (TRAPS).

Optionally the autoimmune disease is selected from the group consisting of any of the types and subtypes of any of multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, and Sjogren's syndrome.

According to at least some embodiments there is provided a method for treating or preventing an infectious disease comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition.

Optionally the infectious disease is selected from the disease caused by bacterial infection, viral infection, fungal infection and/or other parasite infection.

Optionally the infectious disease is selected from hepatitis B, hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

According to at least some embodiments, there is provided a method for treating or preventing cancer comprising administering to a subject in need thereof an effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition.

Optionally the treatment is combined with another moiety or therapy useful for treating cancer.

Optionally the therapy is radiation therapy, antibody therapy, chemotherapy, photodynamic therapy, adoptive T cell therapy, Treg depletion, surgery or in combination therapy with conventional drugs.

Optionally the moiety is selected from the group consisting of immunosuppressants, cytotoxic drugs, tumor vaccines, antibodies (e.g. bevacizumab, erbitux), peptides, pepti-bodies, small molecules, chemotherapeutic agents such as cytotoxic and cytostatic agents (e.g. paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, carboplatin), immunological modifiers such as interferons and interleukins, immunostimulatory antibodies, growth hormones or other cytokines, folic acid, vitamins, minerals, aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, and proteasome inhibitors.

Optionally the cancer is selected from a group consisting of breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), fibrosarcomas and rhabdomyosarcomas, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, keratoacanthomas, renal cancer, anaplastic large-cell lymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer, myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL), and wherein the cancer is non-metastatic, invasive or metastatic.

Optionally the cancer is any of melanoma, cancer of liver, renal, brain, breast, colon, lung, ovary, pancreas, prostate, stomach, multiple myeloma, Hodgkin's lymphoma, non Hodgkin's lymphoma, acute and chronic lymphoblastic leukemia and acute and chronic myeloid leukemia.

According to at least some embodiments, there is provided a method for potentiating a secondary immune response to an antigen in a patient, which method comprises administering effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition.

Optionally the antigen is a cancer antigen, a viral antigen or a bacterial antigen, and the patient has received treatment with an anticancer vaccine or a viral vaccine.

A method of immunotherapy in a patient, comprising:

in vivo or ex vivo tolerance induction, comprising administering effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells;

ex- vivo enrichment and expansion of said cells;

reinfusion of the tolerogenic regulatory cells to said patient.

A method of using at least one of: any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition; as a cancer vaccine adjuvant, comprising administration to a patient an immunogenic amount of a tumor associated antigen preparation of interest; and a cancer vaccine adjuvant in a formulation suitable for immunization, wherein the immune response against the tumor associated antigen in the presence of the cancer vaccine adjuvant is stronger than in the absence of the cancer vaccine adjuvant.

According to at least some embodiments there is provided a method for combining therapeutic vaccination with an antigen along with administration of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition, for treatment of infection.

According to at least some embodiments, there is provided a method for combining any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition, an adjuvant, and an antigen in a vaccine, in order to increase the immune response.

Optionally the antigen is a viral antigen, bacterial antigen, fungal antigen, parasite antigen, and/or other pathogen's antigen.

According to at least some embodiments, any one of the foregoing therapeutic agents according to at least some embodiments of the present invention, including

antibodies and antigen binding fragments and conjugates containing same, and/or alternative scaffolds, against any one of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins; LY6G6F, VSIGIO, TMEM25 and/or LSR secreted or soluble form or ECD and/or variants, and/or orthologs, and/or conjugates thereof, can be used for_adoptive immunotherapy. Immune tolerance or immunological tolerance is the process by which the immune system does not attack an antigen. It can be either 'natural' or 'self tolerance', where the body does not mount an immune response to self antigens, or 'induced tolerance', where tolerance to external antigens can be created by manipulating the immune system. It occurs in three forms: central tolerance, peripheral tolerance and acquired tolerance. Without wishing to be bound by a single theory, tolerance employs regulatory immune cells - including Tregs - that directly suppress autoreactive cells, as well as several other immune cell subsets with immunoregulatory properties - including CD8+ T cells and other types of CD4+ T cells (Trl, Th3), in addition to natural killer (NK), NKT cells, dendritic cells (DC) and B cells.

Tolerance can be induced by blocking costimulation or upon engagement of a co-inhibitory B7 with its counter receptor. Transfer of tolerance involves isolation of the cells that have been induced for tolerance either in vivo (i.e. prior to cell isolation) or ex-vivo, enrichment and expansion of these cells ex vivo, followed by reinfusion of the expanded cells to the patient. This method can be used for treatment of autoimmune diseases as recited herein, immune related diseases as recited herein, transplantation and graft rejection. Thus, according to at least some embodiments, the invention provides methods for tolerance induction, comprising in vivo or ex vivo treatment administration of effective amount of any one of isolated soluble LY6G6F, VSIGIO, TMEM25, LSR polypeptide, or a polypeptide comprising the extracellular domain of LY6G6F, VSIGIO, TMEM25, LSR, or fragment thereof, or a fusion thereof to a heterologous sequence, and/or a polyclonal or monoclonal antibody or antigen binding fragments and conjugates containing same, and/or alternative scaffolds, specific to any one of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells, followed by ex- vivo enrichment and expansion of said cells and reinfusion of the tolerogenic regulatory cells to said patient.

According to at least some embodiments, the invention provides assays for detecting the presence of LY6G6F, VSIG10, TMEM25 and/or LSR proteins in vitro or in vivo in a biological sample or an individual, comprising contacting the sample with an antibody and/or antigen binding fragments and/or conjugates containing same, and/or alternative scaffolds, having specificity for LY6G6F, VSIG10, TMEM25 and/or LSR polypeptides, and detecting the binding of LY6G6F, VSIG10, TMEM25 and/or LSR protein in the sample and/or in the individual.

According to at least some embodiments, there is provided an assay for detecting the presence of any one of the polypeptides of any of SEQ ID NOs:l-8, 11-18, 47-50, 58, 143, or a variant thereof that is at least 95% identical thereto, in a sample.

According to at least some embodiments, there is provided a method for diagnosing a disease in a subject, comprising detecting in the subject or in a sample obtained from said subject any one of the polypeptides of any of SEQ ID NOs: l-8, 11-18, 47-50, 58, 143, or a variant thereof that is at least 95% identical thereto, or fragments thereof.

Optionally detecting the polypeptide is performed in vivo or in vitro.

Optionally the detection is conducted by immunoassay.

Optionally the detection is conducted using antibodies or fragments as described herein.

According to at least some embodiments, the invention provides methods for detecting a disease, diagnosing a disease, monitoring disease progression or treatment efficacy or relapse of a disease, or selecting a therapy for a disease, detect cells affected by the foregoing disease, comprising detecting expression of a LY6G6F, VSIG10, TMEM25 and/or LSR, wherein the disease is selected from cancer, infectious disorder as recited herein, and/or immune related disorder.

According to one embodiment, detecting the presence of the polypeptide is indicative of the presence of the disease and/or its severity and/or its progress. According to another embodiment, a change in the expression and/or the level of the polypeptide compared to its expression and/or level in a healthy subject or a sample obtained therefrom is indicative of the presence of the disease and/or its severity and/or its progress. According to a further embodiment, a change in the expression and/or level of the polypeptide compared to its level and/or expression in said subject or in a sample obtained therefrom at earlier stage is indicative of the progress of the disease. According to still further embodiment, detecting the presence and/or relative change in the

expression and/or level of the polypeptide is useful for selecting a treatment and/or monitoring a treatment of the disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 presents amino acid sequences of LY6G6F (Figure 1A, SEQ ID NO:l),

VSIG10 (Figures IB, SEQ ID NO:3, and 1C, SEQ ID NO:5), TMEM25 (Figures ID, SEQ ID NO:7), LSR (Figures IE (SEQ ID NO: 11), IF (SEQ ID NO: 13), 1G (SEQ ID NO:15), 1H (SEQ ID NO: 16), II (SEQ ID NO: 17), and 1J (SEQ ID NO: 18)) proteins, fragments, ECDs and the corresponding nucleic acid sequences encoding same. Amino acid residues corresponding to signal peptide (SP) appear in bold Italics. Ig-V and/or Ig-C domains are shown in boxes. Amino acid residues corresponding to thransmembrane region (TM) appear in bold and underlined. Amino acid residues corresponding to alternative exons skipping in some of the isoforms (in Figures IB, and IE) appear in Italics and underlined. Nucleic acid sequence corresponding to alternative exons skipping variants of VSIG10 (skiping exon 3), and LSR (isoform-e, skipping exons 3, 4 and 5) appears in bold in Figures 1C, and II, respectievely. Nucleic acid sequence corresponding to transmembrane region (TM) appears in bold and underlined in Figure 1C. Nucleic acid sequence corresponding to signal peptide (SP) appears in bold Italics in Figures 1C, IE, 1G, 1H, II, and 1J. TGA stop codon is highlighted in Figures 1C, and II.

Figure 2 presents amino acid sequence comparison between: the VSIG10 variant SEQ ID NO: 5 and the known VSIG10 protein, SEQ ID NO: 3 (genbank accession number NP_061959.2) (Figure 2A); LSR_isoform-a, SEQ ID NO:l l and known LSR protein, genbank accession number NP_991403 SEQ ID NO: 62 (Figure 2B-1); LSR_isoform-a, SEQ ID NO: 11 and known LSR protein, genbank accession number XP_002829104, SEQ ID NO:68 (Figure 2B-2); LSR_isoform-b, SEQ ID NO: 13 and known LSR protein, genbank accession number NP_057009, SEQ ID NO:63 (Figure 2C-1); LSR_isoform-b, SEQ ID NO:13 and known LSR protein, genbank accession number BAC11614, SEQ ID NO:65 (Figure 2C-2); LSR_isoform-c, SEQ ID NO: 15 and known LSR protein, genbank accession number NP_991404, SEQ ID NO:66 (Figure 2D-1); LSR_isoform-c, SEQ ID NO: 15 and known LSR protein, genbank accession number XP_002829105.1, SEQ ID NO:69 (Figure 2D-2); LSR_isoform-d, SEQ ID NO:16 and known LSR protein, genbank accession number NP_991404, SEQ ID NO: 66 (Figure 2E-1); LSR_isoform-d, SEQ ID NO: 16 and known LSR protein, genbank accession number

XP 002829105.1, SEQ ID NO:69 (Figure 2E-2); LSR_isoform-e, SEQ ID NO: 17 and known LSR protein, genbank accession number BAG59226.1, SEQ ID NO:67 (Figure 2F); LSR_isoform-f, SEQ ID NO: 18 and known LSR protein, genbank accession number NP_991403, SEQ ID NO:62 (Figure 2G-1); LSR_isoform-f, SEQ ID NO: 18 and known LSR protein, genbank accession number NP_991404, SEQ ID NO:66 (Figure 2G-2). The sequence of the unique edge portions (unique junction) of the VSIG10 variant (SEQ ID NO:5) and LSR variant (SEQ ID NO: 18) are bold and highlighted (Figures 2A and 2G, respectively).

Figure 3 shows a scatter plot, demonstrating the expression of VSIG10 transcripts, that encode the VSIG10 proteins, on a virtual panel of all tissues and conditions using MED discovery engine, demonstrating differential expression of VSIG10 transcripts in several groups of cells from the immune system, mainly in leukocytes, and in various cancer conditions, such as CD10+ leukocytes from ALL and BM-CD34+cells from AML.

Figure 4 shows a scatter plot, demonstrating the expression of LSR transcripts, that encode the LSR proteins, on a virtual panel of all tissues and conditions using MED discovery engine, demonstrating differential expression of LSR transcripts in several groups of cells from the immune system, mainly in bone marrow cells, and in various cancerous conditions of tissues, such as in breast, lung, ovary, pancreas, prostate and skin cancers.

Figure 5 A presents LY6G6F human (SEQ ID NO: 1) and mouse (reflNP_001156664.1, SEQ ID NO:29) amino acid sequence comparison. Figure 5B presents VSIG10 human (SEQ ID NO: 3) and mouse (splD3YX43.2, SEQ ID NO:30) amino acid sequence comparison. Figure 5C presents LSR human (SEQ ID NO: 11) and either mouse (reflNP_059101.1, SEQ ID NO:31) or mouse (reflNP_001157656.1, SEQ ID NO:32) amino acid sequence comparison. Figure 5D presents TMEM25 human (SEQ ID NO:7) and mouse (ref: lcll4109, SEQ ID NO:28) amino acid sequence comparison.

Figure 6 presents a table summarizing the primers which were used for cloning of LY6G6F transcript fused to EGFP. Gene specific sequences are shown in bold face; the restriction site extensions utilized for cloning purposes are in Italic; and Kozak sequence are underlined.

Figure 7 presents the DNA sequence of LY6G6F full length_fused to EGFP. The gene specific sequence corresponding to the LY6G6F full length sequence is marked in bold faced, EGFP sequence is unbold Italic underline.

Figure 8 presents the amino acid sequence of the resulting LY6G6F full length fused to EGFP. The gene specific sequence corresponding to the full length sequence of LY6G6F is marked in bold faced; EGFP sequence is unbold Italic underline.

Figure 9 presents cell localization of G6F_EGFP fusion protein transiently expressied in HEK293T cells. The image was obtained using the 40x objective of the confocal microscope.

Figure 10 presents mouse ECDs fused to mouse IgG2a Fc as follows: mouse

LY6G6F (also referred to herein as LY6G6F-Ig, Figure 10A), mouse VSIG10 (Figure 10B), mouse TMEM25 (also referred to herein as TMEM25-Ig, Figure IOC) or mouse LSR (also referred to herein as LSR-Ig, Figure 10D) ECD-mIgG2aFc fused proteins (SEQ ID NOs: 23, 24, 25, or 26, respectively). Amino acid residues corresponding to signal peptide (SP) are shown in Italics. Amino acid residues corresponding to ECD sequence are underlined. Amino acid residues corresponding to mouse IgG2a Fc are shown in bold face (SEQ ID NO:27).

Figure 11 presents amino acid sequences of human ECDs fused to human IgGl Fc with the Cys at position 220 (according to full length human IgGl, position 5 in SEQ ID NO:70) replaced with a Ser (SEQ ID NO: 156), as follows: human LY6G6F (Figure 11 A), human VSIG10 (Figure 11B), human VSIGlO-skipping exon 3 variant (Figure 11C), human TMEM25 (Figure 11D), human LSR isoform a (Figure HE), human LSR isoform b (Figure 11F), human LSR isoform c (Figure 11G), human LSR isoform d (Figure 11H), human LSR isoform e (Figure 111), human LSR isoform f (Figure 11 J) ECD fused to human IgGl Fc (SEQ ID NOs: 71-80, respectively). Amino acid residues corresponding to signal peptide (SP) are shown in bold Italics. Amino acid residues corresponding to human ECD sequence are underlined. Amino acid residues corresponding to human IgGl Fc with the Cys at position 220 replaced with a Ser (SEQ ID NO: 156) are unmarked.

Figure 12 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in cancerous ovary samples relative to the normal samples.

Figure 13 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in cancerous breast samples relative to the normal samples.

Figure 14 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in cancerous lung samples relative to the normal samples.

Figure 15 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in normal tissue samples relative to the ovary samples.

Figure 16 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in cancerous kidney samples relative to the normal samples.

Figure 17 is a histogram showing over expression of the LSR transcripts detectable by or according to LSR_seg24-36_200-309/310_Amplicon (SEQ ID: 140) in cancerous liver samples relative to the normal samples.

Figure 18 demonstrates Western Blot analysis of the expression of LSR_P5a_Flag_m protein (SEQ ID: 144) in stably-transfected recombinant HEK293T cells, as detected with anti Flag (Sigma cat#A8592) (Figure 18 A) and anti LSR antibodies as follow: Abnova, cat#H00051599-B01P (Figure 18B) Abeam, cat ab59646 (Figure 18C) and Sigma cat# HPA007270 (Figure 18D). Lane 1 : HEK293T_pIRESpuro3; lane 2: HEK293T_pIRESpuro3_LSR_P5a_Flag.

Figure 19 demonstrates the subcellular localization of LSR_P5a_Flag_m. LSR_P5a_Flag_m (SEQ ID NO: 144) is localized mainly to the cell cytoplasm, but can also be detected on the cell surface as detected with anti Flag (Sigma cat# A9594) (Figure 19A) and anti LSR antibodies as follows: Abeam, cat ab59646 (Figure 19B) Abnova, cat#H00051599-B01P (Figure 19C) and Sigma cat# HPA007270 (Figurel9D).

Figure 20 demonstrates the endogenous expression of LSR in various cell lines. A band at 72 kDa corresponding to LSR was detected with anti LSR antibody in extracts of (1) Caov3, (2) ES2, (3) OV-90, (4) OVCAR3, (5) SK-OV3 , (6) TOV112D, (7) CaCo2, (8) HeLa, (9) Hep G2, (10) MCF-7, (11) SkBR3 and (12)

293T_LSR_P5a_Flag (Figure 20A). Anti GAPDH (Abeam cat# ab9484) served as a loading control (Figure 20B).

Figure 21 is a histogram showing expression of TMEM25 transcripts detectable by or according to seg21-27 - TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) in normal and cancerous Breast tissues.

Figure 22 is a histogram showing expression of TMEM25 transcripts detectable by or according to seg21-27 - TMEM25_seg_21-27_200-344/346_Amplicon (SEQ ID NO: 123) in different normal tissues.

Figure 23 demonstrates Western blot results showing (A) specific interaction between Rabbit anti TMEM25 antibodies and TMEM25_P5 protein (SEQ ID NO: 7) and TMEM25_P5_Flag (SEQ ID NO: 129), but not HEK_293T_pRp3. (B) specific interaction between TMEM25_P5_Flag protein (SEQ ID NO: 129) and anti-Flag antibodies.Lanel : HEK293T_pIRESpuro3; lane 2: HEK293T_pIRESpuro3_TMEM25-P5, lane 3: HEK293T_pIRESpuro3_TMEM25-P5-Flag.

Figure 24 presents the cell surface localization of TMEM25_P5 (SEQ ID NO : 132)

(Figure 24 A) and TMEM25_P5_Flag (SEQ ID NO: 129) (Figure 24B) using anti TMEM25 Abs. Figure 24C demonstrate TMEM25_P5_Flag (SEQ ID NO: 129) localization using anti flag Abs (Sigma, catalog number: A9594).

Figure 25 demonstrates that anti TMEM25 antibodies bind to the full length TMEM25 protein, in HEK293T recombinant cells expressing TMEM25_P5_Flag protein (1 :2250) (Figure 25 A), as compared to mouse serum (1 :2250) (Figure 25B) used as a negative control, indicating membrane localization of TMEM25 protein.

Figure 26 presents Western Blot results showing the expression of endogenous TMEM25 protein in various cell lines: (1): HEK293T_pIRESpuro3, (2) HEK293T_pIRESpuro3_TMEM25-P5-Flag, (3) KARPAS, (4) G-361, (5) RPMI8226, (6) DAUDI, (7) Jurkat.

Figure 27 demonstrates specific knockdown of TMEM25_P5_Flag protein (SEQ ID NO: 129) in HEK293T cells stably expressing TMEM25_P5_Flag (SEQ ID NO 129) transfected with TMEM25_P5 siRNA (L-018183-00-0005, Dharmacon) (Lane 2) compared to HEK293T cells stably expressing TMEM25_P5_FLAG transfected with

Scrambled-SiRNA (Lane 1) (Dharmacon, D-001810-10-05), using anti TMEM25 antibodies (Sigma, cat# HPA012163).

Figure 28 demonstrates that anti LSR (Cat no. ab59646, Abeam) in sections of positive control cell line (LSR_P5a_Flag_m transfected HEK293T cells (coloumn 1, panels A, C and E) shows specific immunoreactivity in a dose dependent concentrations of 3, 1 and 0.3 ug/ml respectively, as compared to the negative control cell line empty vector HEK293T cells (coloumn 2, panels B, D and F), in pH 9 antigen retrieval method.

Figure 29 demonstrates that anti TMEM25 (Cat no. HPA012163, Sigma) in sections of positive control cell line TMEM25_P5_Flag transfected HEK293T cells (coloumn 1, panels A, C and E) shows specific immunoreactivity in a dose dependent concentrations of 3, 1 and 0.3 ug/ml respectively, as compared to the negative control cell line empty vector HEK293T cells (coloumn 2 panels B, D and F), in pH 9 antigen retrieval method.

Figure 30A-F shows the in vitro inhibitory effect of soluble LY6G6F-Ig (SEQ ID NO:23), TMEM25-Ig (SEQ ID NO:25) and LSR-Ig (SEQ ID NO:26) on mouse T cells activation. Activation of T cells isolated from spleens of DO 11.10 mice was induced with 20ug/ml (Figures 30A-C, E) or 2 ug/ml (Figures D and F) OVA323-339 in the presence of irradiated splenocyted from Balb/c mice that serve as APCs. In these studies CTLA4-Ig or B7-H4-Ig were used as positive controls while mouse IgG2a was used as Ig control.

Figure 31 shows the in vitro inhibitory effect of bead bound LSR-Ig (SEQ ID NO:26) on T cell proliferation induced by anti-CD3 and anti-CD28 coated beads.

Figure 32 shows the effect of LY6G6F, VSIG10, TMEM25 and LSR fusion proteins (SEQ ID NO:23-26, respectively) on CD4 T cell activation, as manifested by reduced IFNy secretion (A) and reduced expression of the activation marker CD69 (B). Each bar is the mean of duplicate cultures, the error bars indicating the standard deviation (Student t-test,*P<0.05, **p<0.01, compared with control mIgG2a.

Figure 33 shows the effect of stimulator cells (a murine thymoma cell line, Bw5147, which were engineered to express membrane-bound anti-human CD3 antibody fragments) expressing the cDNAs encoding human LY6G6F, TMEM25 or LSR (SEQ ID NOs: 1, 7 or 11, respectively) on the proliferation (CPM) of bulk human T cells (Figure 33A), CD4+ human T cells (Figure 33B), CD8+ human T cells (Figure 33C), or naive CD4CD45RA+ human T cells (Figure 33D). Results are displayed as the mean +/- SEM of 6 (Figure 337A) or 3 (Figure 33B, C, and D) experiments. *P<0.05, **p<0.01, ***p<0.001, and #p<0.0001 (Students T-test) represent significantly different results compared to empty vector.

Figure 34 shows the therapeutic effect of LSR-Ig (SEQ ID NO:26) or TMEM25-Ig (SEQ ID NO:25) treatment in the PLP139-151-induced R-EAE model in SJL mice. LSR-Ig (SEQ ID NO:26) or TMEM25-Ig (SEQ ID NO:25) were administered in a therapeutic mode from the onset of disease remission (day 18), at 100 microg/mouse i.p. 3 times per week for two weeks. Therapeutic effects of LSR-Ig and TMEM25-Ig on clinical symptoms are demonstrated as reduction in Mean Clinical Score (Figure 34A). In addition, LSR-Ig and TMEM25-Ig treatment inhibited DTH responses to inducing epitope (PLP139-151) or spread epitope (PLP178-191), on day 35 after R-EAE induction (Figure 34B). In this study the effect of LSR-Ig or TMEM25-Ig was studied in comparison to mIgG2a Ig negative control and CTLA4-Ig positive controlthat were administered at a similar regimen as the test proteins.

Figure 35 shows the dose dependency and mode of action of the effect of TMEM25-Ig (SEQ ID NO:25) in the R-EAE model in SJL mice. In this study, treatments were given from onset of disease remission (day 19) at 100, 30 or 10 microg/mouse i.p. 3 times per week for two weeks, as compared to 100 microg/mouse IgG2a control that was given at a similar schedule, shown are effects of TMEM25-Ig treatment on disease course (Figure 35A), DTH responses to spread epitopes PLP178-191 and MBP84-104 on days 45 and 76 post R-EAE induction (Figure 35B), ex-vivo recall responses of splenocytes isolated on day 45 and 75 post disease induction (Figure 35C) and LN cells isolated on day 45 post disease induction (Figure 35D) as manifested by the effect of TMEM25-Ig treatment on cell proliferation and cytokine secretion (IFNg, IL-17, IL-10 and IL-4). The effect of TMEM25-Ig on cell counts in the spleen, lymph nodes and CNS as well as the different linages present in the CNS upon treatment with TMEM25-Ig at lOOug/dose is shown in Figure 35E.

Figure 36 shows the therapeutic effect of VSIGIO-Ig (SEQ ID NO:24) treatment in the PLP139-151-induced R-EAE model in SJL mice. VSIGIO-Ig (SEQ ID NO:24) was administered in a therapeutic mode from the onset of disease remission (day 19), at 100 microg/mouse i.p. 3 times per week for two weeks. Therapeutic effects of VSIGIO-Ig on clinical symptoms is demonstrated as reduction in Mean Clinical Score (Figure 36A). In addition, VSIGIO-Ig treatment inhibited DTH responses to spread epitopes (PLP178-191 and MBP MBP84-104), on days 45 and 76 after R-EAE induction (Figure 36B). Also shown is the effect of VSIGIO-Ig on ex-vivo recall responses of splenocytes isolated on day 45 and 75 post disease induction (Figure 36C) and LN cells isolated on day 45 post disease induction (Figure 36D) as manifested by the effect of VSIGIO-Ig treatment on cell proliferation and cytokine secretion (IFNg, IL-17, IL-10 and IL-4). The effect of VSIGIO-Ig on cell counts in the spleen, lymph nodes and CNS as well as the different linages present within each of these tissues upon treatment with VSIGIO-Ig at lOOug/dose is shown in Figure 36E. In this study the effect of VSIGIO-Ig was studied in comparison to mIgG2a Ig control that was administered at similar dose and regimen as VSIGIO-Ig.

Figure 37 shows the therapeutic effect of LSR-Ig (SEQ ID NO:26) administred at 100 microg/mouse, i.p, 3 times per week for 10 days in collagen induced arthritis (CIA) model of Rhematoid Arthritis. Measured are clinical score (A) paw swelling (B) and histological damage (C) CTLA4-Ig, (lOOmicrog/mouse) and TNFR-Ig (etanercept) were used as a positive control while mIgG2a Ig control (lOOmicrog/mouse) was used as negative control.

Figure 38 shows the therapeutic effect of LY6G6F-Ig (SEQ ID NO:23) administred at 25mg/kg, i.p, 3 times per week for 2 weeks in collagen induced arthritis (CIA) model of Rhematoid Arthritis, with measurements given according to clinical scores.

For Figures 12-17, 21, 22, division was made into separate parts "A", "B" and so forth for reasons of space only, so as to be able to show all results.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in at least some embodiments, relates to any one of the proteins referred to as LY6G6F, VSIG10, TMEM25 and/or LSR, and its corresponding nucleic acid sequence, and portions and variants thereof and fusion proteins and conjugates containing, and/or polyclonal and monoclonal antibodies and/or antigen binding fragments and/or conjugates containing same, and/or alternative scaffolds thereof that bind LY6G6F, VSIG10, TMEM25 and/or LSR and/or portions and/or variants thereof, and the use thereof as a therapeutic and/or diagnostic agent, and various uses as described herein.

US Patent Application Nos. US2009117566, US20090017473, and other family members, assigned to GENENTECH INC., disclose a 382 amino acid LY6G6F protein sequence (DNA234441, tumor-associated antigenic target (TAT) TAT201, SEQ ID NO:92 therein) having a transmembrane domain between residues 234-254 and 354-374. '566, '473, applications and other applications from this patent family disclose that TAT201 is over expressed in colon and rectal cancers. PCT Application Nos WO2003083074 and WO2004046342 disclose a 382 amino acid LY6G6F protein sequence as one of many genes that are over expressed in colon cancer cells. These patent applications further purportedly relate to methods of use of LY6G6F for detecting and treating colon cancer. However, these patent applications do not teach or suggest or provide any incentive that would direct a skilled artisan to use antibodies specific to the LY6G6F and/or LY6G6F ECD for treatment and/or diagnosis of cancer other than colorectal cancer, and/or infectious disorders, and/or immune related disorders. These patent applications do not describe LY6G6F ECD and do not teach or suggest or provide any incentive that would direct a skilled artisan to use the LY6G6F ECD for treatment of cancer and/or infectious disorders, and/or immune related disorders.

TMEM25 is disclosed in PCT Application Nos W09958642 and WO2003087300, and US Patent Application Nos. US2007041963 and US2005202526, as one of many (hundreds to thousands) proteins, useful for diagnosing, preventing, and treating disorders associated with an abnormal expression or activity of these proteins. However, these applications do not teach or suggest or provide any incentive that would direct a skilled artisan to use antibodies specific to the TMEM25 and/or TMEM25 ECD for treatment and/or diagnosis of cancer and/or infectious disorders, and/or immune related disorders. TMEM25 is also disclosed in US Patent Application No. US2004010134, as one of hundreds of albumin fusion proteins, useful for diagnosing, treating, preventing or ameliorating diseases or disorders e.g. cancer, anemia, arthritis, asthma, inflammatory bowel disease or Alzheimer's disease. However, this application does not teach or suggest or provide any incentive that would direct a skilled artisan to use antibodies specific to the TMEM25 and/or TMEM25 ECD for treatment and/or diagnosis of cancer and/or infectious disorders, and/or immune related disorders. TMEM25 is also discribed in Doolan P, et al., Tumour Biol. 2009, 30(4):200-9 as a favourable prognostic and predictive biomarker for breast cancer diagnosis. However, this publication does not teach or suggest or provide any incentive that would direct a skilled artisan to use the antibodies specific to TMEM25 and/or TMEM25 ECD for treatment of cancer and/or infectious disorders, and/or immune related disorders.

In order that the present invention in various embodiments may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

As used herein the term "isolated" refers to a compound of interest (for example a polynucleotide or a polypeptide) that is in an environment different from that in which the compound naturally occurs e.g. separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature. "Isolated" includes compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or

substantially purified.

An "immune cell" refers to any cell from the hemopoietic origin including but not limited to T cells, B cells, monocytes, dendritic cells, and macrophages.

As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation).

As used herein, a "costimulatory polypeptide" or "costimulatory molecule" is a polypeptide that, upon interaction with a cell-surface molecule on T cells, modulates T cell responses.

As used herein, a "costimulatory signaling" is the signaling activity resulting from the interaction between costimulatory polypeptides on antigen presenting cells and their receptors on T cells during antigen-specific T cell responses. Without wishing to be limited by a single hypothesis, the antigen-specific T cell response is believed to be mediated by two signals: 1) engagement of the T cell Receptor (TCR) with antigenic peptide presented in the context of MHC (signal 1), and 2) a second antigen-independent signal delivered by contact between different costimulatory receptor/ligand pairs (signal 2). Without wishing to be limited by a single hypothesis, this "second signal" is critical in determining the type of T cell response (activation vs inhibition) as well as the strength and duration of that response, and is regulated by both positive and negative signals from costimulatory molecules, such as the B7 family of proteins.

As used herein, the term "B7" polypeptide means a member of the B7 family of proteins that costimulate T cells including, but not limited to B7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3 and biologically active fragments

and/or variants thereof. Representative biologically active fragments include the extracellular domain or fragments of the extracellular domain that costimulate T cells.

As used herein, a "variant" polypeptide contains at least one amino acid sequence alteration as compared to the amino acid sequence of the corresponding wild-type polypeptide.

As used herein, "conservative" amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties. As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant vector can be introduced.

As used herein, the term "an edge portion" or "a new junction"refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein. An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein. A "bridge" may optionally be an edge portion as described above, but may also include a join between a head and a "known protein" portion of a variant, or a join between a tail and a "known protein" portion of a variant, or a join between an insertion and a "known protein" portion of a variant.

In some embodiments, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, or in some embodiments at least about 20 amino acids, or in some embodiments at least about 30 amino acids, or in some embodiments at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant. In some embodiments, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).

It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself.

Furthermore, bridges are described with regard to a sliding window in certain contexts below. For example, certain descriptions of the bridges feature the following format: a bridge between two edges (in which a portion of the known protein is not present in the variant) may optionally be described as follows: a bridge portion of CONTIG-NAME_Pl (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, or at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-NAME_Pl): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for example), in which x varies from 0 to n-2. In this example, it should also be read as including bridges in which n is any number of amino acids between 10-50 amino acids in length. Furthermore, the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1, nor 50 + ((n-2) - x) (for example) greater than the total sequence length.

The term "cancer" as used herein should be understood to encompass any neoplastic disease (whether invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Non-limiting examples of cancer which may be treated with a compound according to at least some embodiments of the present invention are solid tumors, sarcomas and hematological malignancies, including but not limited to breast cancer (e.g. breast carcinoma), cervical cancer, ovary cancer (ovary carcinoma), endometrial cancer, melanoma, bladder cancer (bladder carcinoma), lung cancer (e.g. adenocarcinoma and non-small cell lung cancer), pancreatic cancer (e.g. pancreatic carcinoma such as exocrine pancreatic carcinoma), colon cancer (e.g. colorectal carcinoma, such ascolon adenocarcinoma and colon adenoma), prostate cancer including the advanced disease, hematopoietic tumors of lymphoid lineage (e.g. leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma), myeloid leukemia (for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia), thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanoma, uveal melanoma, teratocarcinoma, neuroblastoma,

glioma, glioblastoma, benign tumor of the skin (e.g. keratoacanthomas), renal cancer, anaplastic large-cell lymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer, myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), and a hereditary cancer syndrome such as Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL), and wherein the cancer may be non-metastatic, invasive or metastatic.

According to at least some preferred embodiments of the present invention, the cancer is selected from the group consisting of melanoma, cancers of liver, renal, brain, breast, colon, lung, ovary, pancreas, prostate, stomach, multiple myeloma and hematopoietic cancer, including but not limited to lymphoma (Hodgkin's and non Hodgkin's), acute and chronic lymphoblastic leukemia and acute and chronic myeloid leukemia, and wherein the cancer may be non-metastatic, invasive or metastatic.

The term "autoimmune disease" as used herein should be understood to encompass any autoimmune disease and chronic inflammatory conditions. According to at least some embodiments of the invention, the autoimmune diseases should be understood to encompass any disease disorder or condition selected from the group including but not limited to multiple sclerosis, including relapsing-remiting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis; psoriasis; rheumatoid arthritis; psoriatic arthritis, systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benign lymphocytic angiitis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic autoimmune hemolytic anemia, pure red cell aplasia, Sjogren's syndrome, rheumatic disease, connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, juvenile rheumatoid arthritis, arthritis uratica, muscular rheumatism, chronic polyarthritis, cryoglobulinemic vasculitis, ANCA-associated vasculitis, antiphospholipid syndrome, myasthenia gravis, autoimmune haemolytic anaemia, Guillian-Barre syndrome, chronic immune

polyneuropathy, autoimmune thyroiditis, insulin dependent diabetes mellitus, type I diabetes, Addison's disease, membranous glomerulonephropathy, Goodpasture's disease, autoimmune gastritis, autoimmune atrophic gastritis, pernicious anaemia, pemphigus, pemphigus vulgarus, cirrhosis, primary biliary cirrhosis, dermatomyositis, polymyositis, fibromyositis, myogelosis, celiac disease, immunoglobulin A nephropathy, Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis, psoriasis, psoriasis arthropathica, Graves' disease, Graves' ophthalmopathy, scleroderma, systemic scleroderma, progressive systemic scleroderma, asthma, allergy, primary biliary cirrhosis, Hashimoto's thyroiditis, primary myxedema, sympathetic ophthalmia, autoimmune uveitis, hepatitis, chronic action hepatitis, collagen diseases, ankylosing spondylitis, periarthritis humeroscapularis, panarteritis nodosa, chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis, chronic urticaria, bullous skin disorders, pemphigoid, atopic eczema, Devic's disease, childhood autoimmune hemolytic anemia, Refractory or chronic Autoimmune Cytopenias, Prevention of development of Autoimmune Anti-Factor VIII Antibodies in Acquired Hemophilia A, Cold Agglutinin Disease, Neuromyelitis Optica, Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne, normocomplementemic urticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler's syndrome, autoimmune retinopathy, age-related macular degeneration, atherosclerosis, chronic prostatitis and TNF receptor-associated periodic syndrome (TRAPS).

Optionally and preferably, the autoimmune disease includes but is not limited to any of the types and subtypes of any of multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, uveitis, or Sjogren's syndrome.

As used herein, "multiple sclerosis" comprises one or more of multiple sclerosis, benign multiple sclerosis, relapsing remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, chronic progressive multiple sclerosis, transitional/progressive multiple sclerosis, rapidly worsening multiple sclerosis, clinically-definite multiple sclerosis, malignant multiple sclerosis, also known as Marburg's Variant, and acute multiple sclerosis. Optionally, "conditions relating to multiple sclerosis" include, e.g., Devic's disease, also known as Neuromyelitis Optica; acute disseminated encephalomyelitis, acute demyelinating optic neuritis, demyelinative transverse myelitis, Miller-Fisher syndrome, encephalomyelradiculoneuropathy, acute demyelinative polyneuropathy, tumef active multiple sclerosis and Balo's concentric sclerosis.

As used herein, "rheumatoid arthritis" comprises one or more of rheumatoid arthritis, gout and pseudo-gout, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Still's disease, ankylosing spondylitis, rheumatoid vasculitis. Optionally, conditions relating to rheumatoid arthritis include, e.g., osteoarthritis, sarcoidosis, Henoch-Schonlein purpura, Psoriatic arthritis, Reactive arthritis, Spondyloarthropathy, septic arthritis, Haemochromatosis, Hepatitis, vasculitis, Wegener's granulomatosis, Lyme disease, Familial Mediterranean fever, Hyperimmunoglobulinemia D with recurrent fever, TNF receptor associated periodic syndrome, and Enteropathic arthritis associated with inflammatory bowel disease.

As used herein, "Uveitis" comprises one or more of uveitis, anterior uveitis (or iridocyclitis), intermediate uveitis (pars planitis), posterior uveitis (or chorioretinitis) and the panuveitic form.

As used herein, "inflammatory bowel disease" comprises one or more of inflammatory bowel disease Crohn's disease, ulcerative colitis (UC), Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's disease, Indeterminate colitis.

As used herein, "psoriasis" comprises one or more of psoriasis, Nonpustular Psoriasis including Psoriasis vulgaris and Psoriatic erythroderma (erythrodermic psoriasis), Pustular psoriasis including Generalized pustular psoriasis (pustular psoriasis of von Zumbusch), Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis, pustular psoriasis of the Barber type, pustular psoriasis of the extremities), Annular

pustular psoriasis, Acrodermatitis continua, Impetigo herpetiformis. Optionally, conditions relating to psoriasis include, e.g., drug-induced psoriasis, Inverse psoriasis, Napkin psoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis, Psoriatic arthritis.

As used herein, "type 1 diabetes" comprises one or more of type 1 diabetes, insulin-dependent diabetes mellitus, idiopathic diabetes, juvenile type Idiabetes, maturity onset diabetes of the young, latent autoimmune diabetes in adults, gestational diabetes. Conditions relating to type 1 diabetes include, neuropathy including polyneuropathy, mononeuropathy, peripheral neuropathy and autonomicneuropathy; eye complications: glaucoma, cataracts, retinopathy.

As used herein, "Sjogren's syndrome" comprises one or more of Sjogren's syndrome, Primary Sjogren's syndrome and Secondary Sjogren's syndrome, as well as conditions relating to Sjogren's syndrome including connective tissue disease, such as rheumatoid arthritis, systemic lupus erythematosus, or scleroderma. Other complications include pneumonia, pulmonary fibrosis, interstitial nephritis, inflammation of the tissue around the kidney's filters, glomerulonephritis, renal tubular acidosis, carpal tunnel syndrome, peripheral neuropathy, cranial neuropathy, primary biliary cirrhosis (PBC), cirrhosis, Inflammation in the esophagus, stomach, pancreas, and liver (including hepatitis), Polymyositis, Raynaud's phenomenon, Vasculitis, Autoimmune thyroid problems, lymphoma.

As used herein, "systemic lupus erythematosus", comprises one or more of systemic lupus erythematosus, discoid lupus, lupus arthritis, lupus pneumonitis, lupus nephritis. Conditions relating to systemic lupus erythematosus include osteo articular tuberculosis, antiphospholipid antibody syndrome, inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis, Lung and pleura inflammation, pleuritis, pleural effusion, chronic diffuse interstitial lung disease, pulmonary

hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome, lupus headache, Guillain-Barre syndrome, aseptic meningitis, demyelinating syndrome, mononeuropathy, mononeuritis multiplex, myasthenia gravis, myelopathy, cranial neuropathy, polyneuropathy, vasculitis.

The term "immune related disease (or disorder or condition)" as used herein should be understood to encompass any disease disorder or condition selected from the group including but not limited to autoimmune diseases, inflammatory disorders and immune disorders associated with graft transplantation rejection, such as acute and chronic rejection of organ transplantation, allogenic stem cell transplantation, autologous stem cell transplantation, bone marrow tranplantation, and graft versus host disease.

As used herein the term "inflammatory disorders" and/or "inflammation", used interchangeably, includes inflammatory abnormalities characterized by disregulated immune response to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammatory disorders underlie a vast variety of human diseases. Non-immune diseases with etiological origins in inflammatory processes include cancer, atherosclerosis, and ischaemic heart disease. Examples of disorders associated with inflammation include: Chronic prostatitis, Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease, Reperfusion injury, Sarcoidosis, Vasculitis, Interstitial cystitis, normocomplementemic urticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced auto-inflammatory syndrome, neonatal onset multisystemic inflammatory disease, familial Mediterranean fever, chronic infantile neurologic, cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler's syndrome, TNF receptor-associated periodic syndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, and inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

As used herein the term "infectious disorder and/or disease" and/or "infection", used interchangeably, includes any disorder, disease and/or condition caused by presence and/or growth of pathogenic biological agent in an individual host organism. As used herein the term "infection" comprises the disorder, disease and/or condition as above, exhibiting clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) and/or which is asymtomatic for much or all of it course. As used herein the term "infection" also comprises disorder, disease and/or condition caused by persistence of foreign antigen that lead to exhaustion T cell phenotype characterized by impaired functionality which is manifested as reduced proliferation and cytokine production. As used herein the term "infectious disorder and/or disease" and/or "infection", further includes any of the below listed infectious disorders, diseases and/or conditions, caused by a bacterial infection, viral infection, fungal infection and /or parasite infection.

As used herein the term "viral infection" comprises any infection caused by a virus, optionally including but not limited to Retro viridae (e.g., human immunodeficiency viruses, such as HIV-1 or HIV-2, acquired immune deficiency (AIDS) also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever virus); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herperviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola virsues, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitides (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1— internally transmitted; class 2— parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses) as well as Severe acute respiratory syndrome virus and respiratory syncytial virus (RSV).

As used herein the term "fungal infection" comprises any infection caused by a fungi, optionally including but not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

As used herein the term "parasite infection" comprises any infection caused by a parasite, optionally including but not limited to protozoa, such as Amebae, Flagellates, Plasmodium falciparum, Toxoplasma gondii, Ciliates, Coccidia, Microsporidia,

Sporozoa; helminthes, Nematodes (Roundworms), Cestodes (Tapeworms), Trematodes (Flukes), Arthropods, and aberrant proteins known as prions.

An infectious disorder and/or disease caused by bacteria may optionally comprise one or more of Sepsis, septic shock, sinusitis, skin infections, pneumonia, bronchitis, meningitis, Bacterial vaginosis, Urinary tract infection (UCI), Bacterial gastroenteritis, Impetigo and erysipelas, Erysipelas, Cellulitis, anthrax, whooping cough, lyme disease, Brucellosis, enteritis, acute enteritis, Tetanus, diphtheria, Pseudomembranous colitis, Gas gangrene, Acute food poisoning, Anaerobic cellulitis, Nosocomial infections, Diarrhea, Meningitis in infants, Traveller's diarrhea, Hemorrhagic colitis, Hemolytic-uremic syndrome, Tularemia, Peptic ulcer, Gastric and Duodenal ulcers, Legionnaire's Disease, Pontiac fever, Leptospirosis, Listeriosis, Leprosy (Hansen's disease), Tuberculosis, Gonorrhea, Ophthalmia neonatorum, Septic arthritis, Meningococcal disease including meningitis, Waterhouse-Friderichsen syndrome, Pseudomonas infection, Rocky mountain spotted fever, Typhoid fever type salmonellosis, Salmonellosis with gastroenteritis and enterocolitis, Bacillary dysentery/Shigellosis, Coagulase -positive staphylococcal infections: Localized skin infections including Diffuse skin infection (Impetigo), Deep localized infections, Acute infective endocarditis, Septicemia, Necrotizing pneumonia, Toxinoses such as Toxic shock syndrome and Staphylococcal food poisoning, Cystitis, Endometritis, Otitis media, Streptococcal pharyngitis, Scarlet fever, Rheumatic fever, Puerperal fever, Necrotizing fasciitis, Cholera, Plague (including Bubonic plague and Pneumonic plague), as well as any infection caused by a bacteria selected from but not limited to Helicobacter pyloris, Boreliai burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. Intracellulare, M. kansaii, M gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp ., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasturella multicoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomeyces israelii.

Non limiting examples of infectious disorder and/or disease caused by virus is selected from the group consisting of but not limited to acquired immune deficiency (AIDS), West Nile encephalitis, coronavirus infection, rhinovirus infection, influenza, dengue, hemorrhagic fever; an otological infection; severe acute respiratory syndrome (SARS), acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection, (gingivostomatitis in children, tonsillitis & pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (herpes labialis, cold sores), aseptic meningitis, Cytomegalovirus infection, Cytomegalic inclusion disease, Kaposi sarcoma, Castleman disease, primary effusion lymphoma, influenza, measles, encephalitis, postinfectious encephalomyelitis, Mumps, hyperplastic epithelial lesions (common, flat, plantar and anogenital warts, laryngeal papillomas, epidermodysplasia verruciformis), croup, pneumonia, bronchiolitis, Poliomyelitis, Rabies, bronchiolitis, pneumonia, German measles, congenital rubella, Hemorrhagic Fever, Chickenpox, Dengue, Ebola infection, Echovirus infection, EBV infection, Fifth Disease, Filovirus, Flavivirus, Hand, foot & mouth disease, Herpes Zoster Virus (Shingles), Human Papilloma Virus Associated Epidermal Lesions, Lassa Fever, Lymphocytic choriomeningitis, Parainfluenza Virus Infection, Paramyxovirus, Parvovirus B 19 Infection, Picornavirus , Poxviruses infection, Rotavirus diarrhea, Rubella, Rubeola, Varicella, Variola infection.

An infectious disorder and/or disease caused by fungi optionally includes but is not limited to Allergic bronchopulmonary aspergillosis, Aspergilloma, Aspergillosis, Basidiobolomycosis, Blastomycosis, Candidiasis, Chronic pulmonary aspergillosis, Chytridiomycosis, Coccidioidomycosis, Conidiobolomycosis, Covered smut (barley), Cryptococcosis, Dermatophyte, Dermatophytid, Dermatophytosis, Endothrix, Entomopathogenic fungus, Epizootic lymphangitis, Epizootic ulcerative syndrome, Esophageal candidiasis, Exothrix, Fungemia, Histoplasmosis, Lobomycosis, Massospora cicadina, Mycosis, Mycosphaerella fragariae, Myringomycosis, Paracoccidioidomycosis, Pathogenic fungi, Penicilliosis, Thousand cankers disease, Tinea, Zeaspora, Zygomycosis.Non limiting examples of infectious disorder and/or disease caused by parasites is selected from the group consisting of but not limited to Acanthamoeba, Amoebiasis, Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis, Balantidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagas disease, Clonorchiasis, Cochliomyia, Coccidia, Chinese Liver Fluke Cryptosporidiosis, Dientamoebiasis, Diphyllobothriasis, Dioctophyme renalis infection, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Halzoun Syndrome, Isosporiasis, Katayama fever, Leishmaniasis, lymphatic filariasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Primary amoebic meningoencephalitis, Parasitic pneumonia, Paragonimiasis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Sparganosis, Rhinosporidiosis, River blindness, Taeniasis (cause of Cysticercosis), Toxocariasis, Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis, Trypanosomiasis, Tapeworm infection.

A preferred example of infectious disease is a disease caused by any of hepatitis B, hepatitis C, infectious mononucleosis, EBV, cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

As used herein, the term "vaccine" refers to a biological preparation that improves immunity to a particular disease, wherein the vaccine includes an antigen, such as weakened or killed forms of pathogen, its toxins or one of its surface proteins, against which immune responses are elicited. A vaccine typically includes an adjuvant as immune potentiator to stimulate the immune system. As used herein, the term "therapeutic vaccine" and/or "therapeutic vaccination" refers to a vaccine used to treat ongoing disease, such as infectious disease or cancer.

As used herein, the term "adjuvant" refers to an agent used to stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself.

As used herein, the term LY6G6F and/or LY6G6F protein(s) refers to any one of the proteins set forth in SEQ ID NO: l, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding for same, that are differentially expressed in cancers as recited herein and/or in infectious disorders as recited herein, and/or immune related disorders as recited herein, and/or that play a role in the etiology of cancers, and/or in infectious disorders, and/or immune related disorders.

According to preferred embodiments, a LY6G6F fragment comprises an amino acid sequence of LY6G6F ectodomain, set forth in any one of SEQ ID NOs: 2, 59, 81, 96, and/or variants thereof. According to preferred embodiments, a LY6G6F ortholog comprises any one of SEQ ID NOs:20, 29. According to preferred embodiments, a nucleic acid sequence encoding LY6G6F protein comprises SEQ ID NO:33, 57 or 182.

As used herein, the term VSIG10 and/or VSIG10 protein(s) refers to any one of the proteins set forth in any one of SEQ ID NOs:3, 5, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding for same, that are differentially expressed in cancers as recited herein and/or in infectious disorders as recited herein, and/or immune related disorders as recited herein, and/or that play a role in the etiology of cancers and/or in infectious disorders, and/or immune related disorders.

According to preferred embodiments, a VSIG10 fragment comprises an amino acid sequence of VSIG10 ectodomain, set forth in any one of SEQ ID NOs: 4, 6, 60, 61, 82-93, 97-100, and/or variants thereof, and/or an amino acid sequence comprising a VSIG10 variant (SEQ ID NO:5) unique edge portion, demonstrated in Figure 2A. According to preferred embodiments, a VSIG10 ortholog comprises any one of SEQ ID NOs: 19, 30. According to preferred embodiments, a nucleic acid sequence encoding VSIG10 protein comprises any one of SEQ ID NOs: 34, 35, 36, 183, or 184.

As used herein, the term TMEM25 and/or TMEM25 protein(s) refers to any one of the proteins set forth in any one of SEQ ID NOs: 7, 39, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding for same, that are differentially expressed in cancers as recited herein and/or in infectious disorders as recited herein, and/or immune related disorders as recited herein, and/or that play a role in the etiology of cancers and/or in infectious disorders, and/or immune related disorders.

According to preferred embodiments, a TMEM25 fragment comprises an amino acid sequence of TMEM25 ectodomain, set forth in any one of SEQ ID NOs: 8, 39, 94, 101 and/or variants thereof. According to preferred embodiments, a TMEM25 ortholog comprises a protein having a sequence according to any of SEQ ID NO: 9, and/or 28. According to preferred embodiments, a nucleic acid sequence encoding TMEM25 protein comprises any one of SEQ ID NOs:37 or 185.

As used herein, the term LSR and/or LSR protein(s) refers to any one of the proteins set forth in any one of SEQ ID NOs: 11, 13, 15-18, 143, and/or variants thereof, and/or orthologs and/or fragments thereof, and/or nucleic acid sequences encoding for same, that are differentially expressed in cancers as recited herein and/or in infectious disorders as recited herein, and/or immune related disorders as recited herein, and/or that play a role in the etiology of cancers and/or in infectious disorders, and/or immune related disorders.

According to preferred embodiments, a LSR fragment comprises an amino acid sequence of LSR ectodomain, set forth in any one of SEQ ID NOs: 10, 12, 14, 22, 47-50, 95, 102, and/or variants thereof, and/or an amino acid sequence comprising a LSR variant (SEQ ID NO: 18) unique edge portion, demonstrated in Figure 2G. An example of LSR orthologs is presented in any one of SEQ ID NOs: 21, 31, 32. According to preferred embodiments, a nucleic acid sequence encoding LSR protein comprises any one of SEQ ID NOs: 40-46, 132, 155, 188, 186, 187, 145, 154.

Without wishing to be limited by a single hypothesis, each of the LY6G6F, VSIG10, TMEM25 and/or LSR proteins according to at least some embodiments of the present invention, was predicted to be an immune costimulatory protein, e.g., a B7 protein family member that is involved in B7 immune co-stimulation including for example T cell responses elicited against cancer cells and that elicit effects on immunity such as triggering of autoimmune effects.

As used herein, the term the "soluble ectodomain (ECD)" or "ectodomain" or "soluble LY6G6F, VSIG10, TMEM25 and/or LSR protein(s)/molecule(s)" of LY6G6F, VSIG10, TMEM25 and/or LSR means non-cell-surface-bound (i.e. circulating) LY6G6F, VSIG10, TMEM25 and/or LSR molecules or any portion thereof, including, but not limited to: LY6G6F, VSIG10, TMEM25 and/or LSR-Ig fusion proteins, wherein the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR is fused to an immunoglobulin (Ig) moiety rendering the fusion molecule soluble, or fragments and derivatives thereof, proteins with the extracellular domain of LY6G6F, VSIG10, TMEM25 and/or LSR fused or joined with a portion of a biologically active or chemically active protein such as the papillomavirus E7 gene product, melanoma-associated antigen p97 or HIV env protein, or fragments and derivatives thereof; hybrid (chimeric) fusion proteins such as LY6G6F, VSIG10, TMEM25 and/or LSR-Ig, or fragments and derivatives thereof. Such fusion proteins are described in greater detail below.

"Soluble LY6G6F, VSIG10, TMEM25 and/or LSR protein(s)/molecule(s)" also include LY6G6F, VSIG10, TMEM25 and/or LSR molecules with the transmembrane domain removed to render the protein soluble, or fragments and derivatives thereof; fragments, portions or derivatives thereof, and soluble LY6G6F, VSIG10, TMEM25 and/or LSR mutant molecules. The soluble LY6G6F, VSIG10, TMEM25 and/or LSR

molecules used in the methods according to at least some embodiments of the invention may or may not include a signal (leader) peptide sequence.

Fragments of LY6G6F polypeptides

The term the "soluble ectodomain (ECD)" or "ectodomain" or "soluble" form of

LY6G6F refers also to the nucleic acid sequences encoding the corresponding proteins of LY6G6F "soluble ectodomain (ECD)" or "ectodomain" or "soluble LY6G6F proteins/molecules"). Optionally, the LY6G6F ECD refers to any one of the polypeptide sequences below and/or listed in Table A below, and/or or fragments or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or conjugates thereof, and/or polynucleotides encoding same:

SEQ ID NO: 2, amino acid residues 17-234 (not including signal peptide, up till transmembrane) (Figure 1A): ADNMQAI YVALGEA VELPCPSPPTLHGDEHLS WFCSPA AGSFTTLVAQVQ VGRP APDPGKPGRESRLRLLGNYSLWLEGSKEEDAGRYWCAVLGQHHNYQNWRVYD VLVLKGSQLSARAADGSPCNVLLCSVVPSRRMDSVTWQEGKGPVRGRVQSFWG SEAALLLVCPGEGLSEPRSRRPRIIRCLMTHNKGVSFSLAASIDASPALCAPSTGW DMP,

and fragments and variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith. SEQ ID NO:59 represents an example of the LY6G6F ECD including signal peptide.

Table A

Optionally, the fragment is of at least about 62, 63, 64, 65 and so forth amino acids of the extracellular domain of LY6G6F protein, set forth in SEQ ID NO: 1, up to 228 amino acids of the LY6G6F protein extracellular domain, optionally including any integral value between 62 and 228 amino acids in length. Preferably, the fragment is of at least about 62 and up to 82 amino acids of the LY6G6F protein extracellular domain, optionally including any integral value between 62 and 82 amino acids in length. Also preferably the fragment is of at least about 95 up to 115 amino acids of the LY6G6F protein extracellular domain, optionally including any integral value between 95 and 115 amino acids in length. Also preferably the fragment is of at least about 208 up to 228 amino acids of the LY6G6F protein extracellular domain, optionally including any integral value between 208 and 228 amino acids in length. More preferably, the fragment is about 72 or 106 or 218 amino acids. The LY6G6F fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the LY6G6F transmembrane domain.

In particular, the fragments of the extracellular domain of LY6G6F can include any sequence corresponding to any portion of or comprising the IgV domain of the extracellular domain of LY6G6F, having any sequence corresponding to residues of LY6G6F (SEQ ID NO: l) starting from any position between 14 and 27 and ending at any position between 112 and 132.

The LY6G6F proteins contain an immunoglobulin domain within the extracellular domain, the IgV domain (or V domain), shown in Figure 1 A in a box, which is related to the variable domain of antibodies. The IgV domain may be responsible for receptor binding, by analogy to the other B7 family members. The Ig domain of the extracellular domain includes one disulfide bond formed between intradomain cystein residues, as is typical for this fold and may be important for structure-function. In SEQ ID NO: 1 these cysteines are located at residues 35 and 106.

In one embodiment, there is provided a soluble fragment of LY6G6F; as described in greater detail below with regard to the section on fusion proteins, such a soluble fragment may optionally be described as a first fusion partner. Useful fragments are those that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. A LY6G6F polypeptide that is a fragment of full-length LY6G6F typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptor(s) and/or of the ability to inhibit T cell activation as compared to full-length LY6G6F. Soluble LY6G6F polypeptide fragments are fragments of LY6G6F polypeptides that may be shed, secreted or otherwise extracted from the producing cells. In other embodiments, the soluble fragments of LY6G6F polypeptides include fragments of the LY6G6F extracellular domain that retain LY6G6F biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the LY6G6F extracellular domain polypeptide comprises the amino acid sequence of the IgV domain as set forth in any one of SEQ ID NO: 81, or fragments or variants thereof, or the region between the conserved cysteines of the IgV domain located at residues 35 and 106 of the full-length protein SEQ ID NO: l, corresponding to the sequence set forth in SEQ ID NO: 96: CPSPPTLHGDEHLSWFCSPAAGSFTTLVAQVQVGRPAPDPGKPGRESRLRLLGNY SLWLEGS KEED AGR YWC . In other embodiments the LY6G6F extracellular domain polypeptide consists essentially of the amino acid sequence of the IgV domain as set forth in any one of SEQ ID NOs: 81 and 96.

Generally, the LY6G6F polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence of LY6G6F can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. The signal peptide sequence that is used to replace the LY6G6F signal peptide sequence can be any known in the art.

Optionally, the LY6G6F ECD refers to any one of the nucleic acid sequences encoding LY6G6F ECD polypeptides, optionally to the nucleic acid sequences set forth in SEQ ID NO:33, or fragments thereof and/or degenerative variants thereof, encoding LY6G6F ECD polypeptides set forth in SEQ ID NO:2.

Optionally, the LY6G6F ECD refers to orthologous ECD polypeptides. Optionally, the LY6G6F ECD refers to mouse LY6G6F ECD polypeptides, set forth in SEQ ID NOs:20, and/or a mouse LY6G6F ECD-IgG2a-Fc-fused polypeptide, set forth in SEQ ID NOs:23.

Fragments of VSIGIO polypeptides

The term the "soluble ectodomain (ECD)" or "ectodomain" or "soluble" form of VSIGIO refers also to the nucleic acid sequences encoding the corresponding proteins of VSIGIO "soluble ectodomain (ECD)" or "ectodomain" or "soluble VSIGIO proteins/molecules"). Optionally, the VSIGIO ECD refers to any one of the polypeptide sequences below and/or listed in Table B below, and/or fragments or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or conjugates thereof, and/or polynucleotides encoding same:

SEQ ID NO: 4, amino acid residues 31-413 (not including signal peptide, up till transmembrane) (Figure IB):

VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDA TSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVASGPYQIEVHIVATGTLPNGTL YAARGSQVDFSCNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQG NYTCLALNQLSKRHRKVTTELLVYYPPPSAPQCWAQMASGSFMLQLTCRWDGG YPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIVGPESGASCM VQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVIIQPSSRH LITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEPLNIGG;

SEQ ID NO: 6, amino acid residues 31-312 (skipping exon 3 variant, not including signal peptide, up till transmembrane) (Figure 1C):

VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDA TSLHIESLSLGDEGIYTCQEILNVTQWFQVWLQVANPPPSAPQCWAQMASGSFML QLTCRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKCVTSHIV GPESGASCMVQIRGPSLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILWLRNLTQP EVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYICRADSPVGVREMEIWLSVKEP LNIGG,

and variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith. SEQ ID NOs:60-61 represent examples of the VSIGIO ECD including signal peptide.

Table B

DFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKK

FKCVTSHIVGPESGASCMVQIR

90 PYQIEVHIVATGTLPNGTLYAARGSQVDFSCNS VSIG10_WT_IgC2_doraains_2-4 SSRPPPVVEWWFQALNSSSESFGHNLTVNFFSL aa 123-404 of seq id:3 LLISPNLQGNYTCLALNQLSKRHRKVTTELLVY YPPPSAPQCWAQMASGSFMLQLTCRWDGGYPDP DFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKK FKCVTSHIVGPESGASCMVQIRGPSLLSEPMKT CFTGGNVTLTCQVSGAYPPAKILWLRNLTQPEV IIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYY ICRADSPVGVREMEIWLS

91 PPPSAPQCWAQMASGSFMLQLTCRWDGGYPDPD VSIG10_IgC2_doraains_3-4 aa FLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKF 223-404 of seq id:3 aa 122- 303 of seq id : 5

KCVTSHIVGPESGASCMVQIRGPSLLSEPMKTC FTGGNVTLTCQVSGAYPPAKILWLRNLTQPEVI IQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYI CRADSPVGVREMEIWLS

92 VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEP VSIG10_Variant_skipping_exon

VFLLSSNSSLRPAEPRFSLVDATSLHI _3_T95617_P6_IgC2_doraains_l ,

ESLSLGDEGIYTCQEILNVTQWFQVWLQVANPP 3 aa 31-208 of seq id:5

PSAPQCWAQMASGSFMLQLTCRWDGGY

PDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSD

GKKFKCVTSHIVGPESGASCMVQIR

93 VVIGEVHENVTLHCGNISGLRGQVTWYRNNSEP VSIG10_Variant_skipping_exon

VFLLSSNSSLRPAEPRFSLVDATSLHI _3_T95617_P6_IgC2_doraains_l ,

ESLSLGDEGIYTCQEILNVTQWFQVWLQVANPP 3-4 aa 31-303 of seq id: 5

PSAPQCWAQMASGSFMLQLTCRWDGGY

PDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSD

GKKFKCVTSHIVGPESGASCMVQIRGP

SLLSEPMKTCFTGGNVTLTCQVSGAYPPAKILW

LRNLTQPEVIIQPSSRHLITQDGQNST

LTIHNCSQDLDEGYYICRADSPVGVREMEIWLS

Optionally, the fragment is of at least about 36, 37, 38, 39, 40, 41, 42, 43, and so forth amino acids of the extracellular domain of VSIGIO protein, set forth in SEQ ID NO:3, up to 393 amino acids of the VSIGIO protein extracellular domain, optionally, including any integral value between 36 and 393 amino acids in length. Preferably, the fragment is of at least about 36 up to 70 amino acids of the VSIGIO protein extracellular domain, optionally including any integral value between 36 and 70 amino acids in length. Also preferably the fragment is of at least about 80 up to 100 amino acids of the VSIGIO protein extracellular domain, optionally including any integral value between 80 and 100 amino acids in length. Also preferably the fragment is of at least about 170 up to 200 amino acids of the VSIGIO protein extracellular domain, optionally including any integral value between 170 and 200 amino acids in length. Also preferably the fragment is of at least about 265 up to 290 amino acids of the VSIGIO protein extracellular domain, optionally including any integral value between 265 and 290 amino acids in length. Also preferably the fragment is of at least about 365 up to 393 amino acids of the VSIGIO protein extracellular domain, optionally including any integral value between 365 and 393 amino acids in length. More preferably, the fragment is about 46, 49, 58, 60, 87, 89, 93, 94, 178, 182, 185, 187, 273, 279, 282, 374, 383 amino acids. The VSIGIO fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the VSIGIO transmembrane domain.

In particular, the fragments of the extracellular domain of VSIGIO can include any sequence corresponding to any portion of or comprising of one or more of the IgC2 domains of the extracellular domain of VSIGIO, having any sequence corresponding to residues of VSIGIO (SEQ ID NO:3) starting from any position between 28 and 41 and ending at any position between 109 and 122 or starting from any position between 120 and 133 and ending at any position between 205 and 222 or starting from any position between 216 and 233 and ending at any position between 299 and 310 or starting from any position between 310 and 321 and ending at any position between 394 and 414 or starting from any position between 28 and 41 and ending at any position between 205 and 222 or starting from any position between 28 and 41 and ending at any position between 299 and 310 or starting from any position between 28 and 41 and ending at any position between 394 and 414 or starting from any position between 120 and 133 and ending at any position between 299 and 310 or starting from any position between 120 and 133 and ending at any position between 394 and 414 or starting from any position between 216 and 233 and ending at any position between 394 and 414, or having any sequence corresponding to residues of VSIG10_Variant_skipping_exon_3_T95617_P6 (SEQ ID NO:5) starting from any position between 28 and 41 and ending at any position between 198 and 209 or starting from any position between 28 and 41 and ending at any position between 293 and 313.

The VSIGIO proteins contain immunoglobulin domains within the extracellular domain, IgC2 domain (or Ig-like C2 domain or Ig C2-set domain), which is related to the constant domain of antibodies. The domains are illustrated in Figure IB (for SEQ ID NO:3) snd in Figure 1C (for SEQ ID NO:5). The Ig domains of the extracellular domain include one disulfide bond formed between intradomain cystein residues, as is typical for this fold and may be important for structure-function. In SEQ ID NO: 3 these cysteines are located at residues 44 and 103 and at residues 153 and 201 and at residues 245 and 290 and at residues 331 and 388. In SEQ ID NO:5 these cysteines are located at residues 44 and 103 and 144 and 189 and at residues 230 and 287.

In one embodiment, there is provided a soluble fragment of VSIG10, which may optionally be described as a first fusion partner in the below section on fusion proteins. Useful fragments are those that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. A VSIG10 polypeptide that is a fragment of full-length VSIG10 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptor(s) and/or of the ability to inhibit T cell activation as compared to full-length VSIG10. Soluble VSIG10 polypeptide fragments are fragments of VSIG10 polypeptides that may be shed, secreted or otherwise extracted from the producing cells. In other embodiments, the soluble fragments of VSIG10 polypeptides include fragments of the VSIG10 extracellular domain that retain VSIG10 biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the VSIG10 extracellular domain polypeptide comprises the amino acid sequence of at least one of the IgC2 domains as set forth in any one of SEQ IDS NO: 82, 83, 84 and 85, or fragments or variants thereof, or the regions between the conserved cysteines of the IgC2 domains located at residues 44 and 103 of the full-length protein SEQ ID NO:3, corresponding to the sequence set forth in SEQ ID NO: 97: CGNISGLRGQVTWYRNNSEPVFLLSSNSSLRPAEPRFSLVDATSLHIESLSLGDEGI YTC, or located at residues 153 and 201 of the full-length protein SEQ ID NO:3, corresponding to the sequence set forth in SEQ ID NO: 98: CNSSSRPPPVVEWWFQALNSSSESFGHNLTVNFFSLLLISPNLQGNYTC or located at residues 245 and 209 of the full-length protein SEQ ID NO:3, corresponding to the sequence set forth in SEQ ID NO: 99:

CRWDGGYPDPDFLWIEEPGGVIVGKSKLGVEMLSESQLSDGKKFKC or located at residues 331 and 388 of the full-length protein SEQ ID NO:3, corresponding to the sequence set forth in SEQ ID NO: 100:

CQVSGAYPPAKILWLRNLTQPEVIIQPSSRHLITQDGQNSTLTIHNCSQDLDEGYYI

C. In some further embodiments the VSIGIO extracellular domain polypeptide consists essentially of amino acid sequence of at least one of SEQ IDS NOs: 82-93, 97-100.

Generally, the VSIGIO polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence of VSIGIO can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. The signal peptide sequence that is used to replace the VSIGIO signal peptide sequence can be any known in the art.

Optionally, the VSIGIO ECD refers also to any one of the nucleic acid sequences encoding VSIGIO ECD polypeptides, optionally to the nucleic acid sequences set forth in SEQ ID NOs:34, 36, or fragments thereof and/or degenerative variants thereof, encoding VSIGIO ECD polypeptides set forth in SEQ ID NOs:4, 6, respectively.

Optionally, the VSIGIO ECD refers to orthologous ECD polypeptides. Optionally, the VSIGIO ECD refers to mouse VSIGIO ECD polypeptides, set forth in SEQ ID NO: 19, and/or a mouse VSIGIO ECD-IgG2a-Fc-fused polypeptide, set forth in SEQ ID NO:24.

Fragments of TMEM25 polypeptides

The term the "soluble ectodomain (ECD)" or "ectodomain" or "soluble" form of TMEM25 refers also to the nucleic acid sequences encoding the corresponding proteins of TMEM25 "soluble ectodomain (ECD)" or "ectodomain" or "soluble TMEM25 proteins/molecules"). Optionally, the TMEM25 ECD refers to any one of the polypeptide sequences below and/or listed in Table C below, and/or fragments or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or conjugates thereof, and/or polynucleotides encoding same:

SEQ ID NO: 8, amino acid residues 27-232 (not including signal peptide, up till transmembrane) (Figure ID):

ELEPQIDGQTWAERALRENERHAFTCRVAGGPGTPRLAWYLDGQLQEASTSRLL SVGGEAFSGGTSTFTVTAHRAQHELNCSLQDPRSGRSANASVILNVQFKPEIAQV GAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNTSDFLVLDAQNYP WLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRVE,

and variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith. SEQ ID NO:39 represents example of the TMEM25 ECD including signal peptide.

Table C

Optionally, the fragment is of at least about 46, 47, 48, 49, 50, 51, 52, and so forth amino acids of the extracellular domain of TMEM25 protein, set forth in SEQ ID NO:7, up to 216 amino acids of the TMEM25 protein extracellular domain, optionally including any integral value between 46 and 216 amino acids in length. Preferably, the fragment is of at least about 46 up to 66 amino acids of the TMEM25 protein extracellular domain, optionally including any integral value between 46 and 66 amino acids in length. Also preferably the fragment is of at least about 84 up to 104 amino acids of the TMEM25 protein extracellular domain, optionally including any integral value between 84 and 104 amino acids in length. Also preferably the fragment is of at least about 196 up to 216 amino acids of the TMEM25 protein extracellular domain, optionally including any integral value between 196 and 216 amino acids in length. More preferably, the fragment is about 56 or 94 or 206 amino acids. The TMEM25 fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the TMEM25 transmembrane domain.

In particular, the fragments of the extracellular domain of TMEM25 can include any sequence corresponding to any portion of or comprising the IgC2 domain of the extracellular domain of TMEM25, having any sequence corresponding to residues of TMEM25 (SEQ ID NO:7) starting from any position between 27 and 40 and ending at any position between 113 and 133.

The TMEM25 proteins contain an immunoglobulin domain within the extracellular domain, IgC2 domain (or Ig-like C2 domain or Ig C2-set domain), which is related to the constant domain of antibodies. The domain is shown in Figure ID in a box. The Ig domain of the extracellular domain includes one disulfide bond formed between intradomain cystein residues, as is typical for this fold and may be important for structure -function. In SEQ ID NO: 7 these cysteines are located at residues 52 and 107.

In one embodiment, there is provided a soluble fragment of TMEM25, which may optionally be described as a first fusion partner, as for example in the detailed section on fusion proteins below. Useful fragments are those that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. A TMEM25 polypeptide that is a fragment of full-length TMEM25 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptor(s) and/or of the ability to inhibit T cell activation as compared to full-length TMEM25. Soluble TMEM25 polypeptide fragments are fragments of TMEM25 polypeptides that may be shed, secreted or otherwise extracted from the producing cells. In other embodiments, the soluble fragments of TMEM25 polypeptides include fragments of the TMEM25 extracellular domain that retain TMEM25 biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the TMEM25 extracellular domain polypeptide comprises the amino acid sequence of IgC2 domain, as set forth in any one of SEQ ID NO: 94, or fragments or variants thereof, or the region between the conserved cysteines of the IgC2 domain located at residues 52 and 107 of the full-length protein SEQ ID NO:7, corresponding to the sequence set forth in SEQ ID NO: 101 : CRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHEL NC. In other embodiments the TMEM25 extracellular domain polypeptide consists essentially of the amino acid sequence of the IgC2 domain as set forth in any one of SEQ ID NOs: 94 and 101.

Generally, the TMEM25 polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence of TMEM25 can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. The signal peptide sequence that is used to replace the TMEM25 signal peptide sequence can be any known in the art.

Optionally, the TMEM25 ECD refers also to any one of the nucleic acid sequences encoding TMEM25 ECD polypeptides, optionally to the nucleic acid sequences set forth in SEQ ID NO: 37, or fragments thereof and/or degenerative variants thereof, encoding TMEM25 ECD polypeptides set forth in SEQ ID NO: 8

Optionally, the TMEM25 ECD refers to orthologous ECD polypeptides. Optionally, the TMEM25 ECD refers to mouse TMEM25 ECD polypeptides, set forth in SEQ ID NOs:9, and/or a mouse TMEM25 ECD-IgG2a-Fc-fused polypeptide, set forth in SEQ ID NOs:25.

Fragments of LSR polypeptides

The term the "soluble ectodomain (ECD)" or "ectodomain" or "soluble" form of LSR refers also to the nucleic acid sequences encoding the corresponding proteins of LSR "soluble ectodomain (ECD)" or "ectodomain" or "soluble LSR proteins/molecules"). Optionally, the LSR ECD refers to any one of the polypeptide sequences below and/or

listed in Table D below, and/or fragments or variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith, and/or conjugates thereof, and/or polynucleotides encoding same:

SEQ ID NO: 12, LSR isoform A ECD (not including signal peptide, up till transmembrane) amino acid residues 42-211 (Figure IE):

IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT GNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPG FQAGPIED;

SEQ ID NO: 14, LSR isoform B ECD (not including signal peptide, up till transmembrane) amino acid residues 42-192 (Figure IF):

IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT GNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLD;

SEQ ID NO: 47, LSR isoform C secreted variant amino acid residues 42-533 (Figure 1G):

IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT GNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSG VPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYV PLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVE RAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGG GWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYD QDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLL EEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESL VV;

SEQ ID NO: 48, LSR isoform D secreted variant amino acid residues 42-532 (Figure 1H)

IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV

DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT

GNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLVYAAGKAATSG VPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDVDRSSSAGGQGSYV PLLRDTDSSVASVRSGYRIQASQQDDSMRVLYYMEKELANFDPSRPGPPSGRVER AMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPRGWDQEPAREQAGGG WRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAYMPPRSRSRDDLYDQ DDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDNGSRSGDLPYDGRLLE EAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASRERRLKKNLALSRESL VV;

SEQ ID NO: 49, LSR isoform E secreted variant amino acid residues 42-493 (Figure II): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT GMYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGGYPGDV DRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYMEKELAN FDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHSPRSPR GWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGGRSRAY MPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTRDPRDN GSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETDSQASR ERRLKKNLALSRESLVV;

SEQ ID NO: 50, LSR isoform F secreted variant amino acid residues 42-552 (Figure 1J): IQVTVSNPYHVVILFQPVTLPCTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASV DNQLNAQLAAGNPGYNPYVECQDSVRTVRVVATKQGNAVTLGDYYQGRRITIT GNADLTFDQTAWGDSGVYYCSVVSAQDLQGNNEAYAELIVLGRTSGVAELLPG FQAGPIEVYAAGKAATSGVPSIYAPSTYAHLSPAKTPPPPAMIPMGPAYNGYPGG YPGDVDRSSSAGGQGSYVPLLRDTDSSVASEVRSGYRIQASQQDDSMRVLYYME KELANFDPSRPGPPSGRVERAMSEVTSLHEDDWRSRPSRGPALTPIRDEEWGGHS PRSPRGWDQEPAREQAGGGWRARRPRARSVDALDDLTPPSTAESGSRSPTSNGG RSRAYMPPRSRSRDDLYDQDDSRDFPRSRDPHYDDFRSRERPPADPRSHHHRTR DPRDNGSRSGDLPYDGRLLEEAVRKKGSEERRRPHKEEEEEAYYPPAPPPYSETD SQASRERRLKKNLALSRESLVV,

and variants thereof possessing at least 80% sequence identity, more preferably at least 90% sequence identity therewith and even more preferably at least 95, 96, 97, 98 or 99% sequence identity therewith. SEQ ID NOs: 10, 22 represent example of the LSR ECD including signal peptide.

Optionally, the fragment is of at least about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 and so forth amino acids of the extracellular domain of LSR protein, set forth in SEQ ID NO: 11 and/or 143, up to 198 amino acids of the extracellular domain, optionally including any integral value between 100 and 198 amino acids in length. The LSR fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the LSR transmembrane domain.

Table D

Optionally, the fragment is of at least about 98, 99, 100, 101, 102 and so forth amino acids of the extracellular domain of LSR protein, set forth in SEQ ID NO: 11, up to 180 amino acids of the LY6G6F protein extracellular domain, optionally including any integral value between 98 and 180 amino acids in length Preferably, the fragment is of at least about 98 up to 118 amino acids of the LSR protein extracellular domain, optionally including any integral value between 98 and 118 amino acids in length. Also preferably the fragment is of at least about 135 up to 155 amino acids of the LSR protein extracellular domain, optionally including any integral value between 135 and 155 amino acids in length. Also preferably the fragment is of at least about 160 up to 180 amino acids of the LSR protein extracellular domain, optionally including any integral value between 160 and 180 amino acids in length. More preferably, the fragment is about 108 or 145 or 170 amino acids. The LSR fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the LSR transmembrane domain.

The LSR proteins contain an immunoglobulin domain within the extracellular domain, the IgV domain (or V domain), which is related to the variable domain of antibodies. The Ig domain is shown in a box in Figures IE, IF, 1G, 1H, and 1J, for SEQ ID NOs: 11, 13, 15, 16, and 18, respectively. The Ig domain of the extracellular domain includes one disulfide bond formed between intradomain cystein residues, as is typical for this fold and may be important for structure -function. In SEQ ID NO: 11 these cysteines are located at residues 63 and 170.

In one embodiment, there is provided a soluble fragment of LSR, which may optionally be described as a first fusion partner, as for example in the below section on fusion proteins. Useful fragments are those that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. A LSR polypeptide that is a fragment of full-length LSR typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptor(s) and/or of the ability to inhibit T cell activation as compared to full-length LSR. Soluble LSR polypeptide fragments are fragments of LSR polypeptides that may be shed, secreted or otherwise extracted from the producing cells. In other embodiments, the soluble fragments of LSR polypeptides include fragments of the LSR extracellular domain that retain LSR biological activity, such as fragments that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the LSR extracellular domain polypeptide comprises the amino acid of the IgV domain as set forth in any one of SEQ ID NO: 95, or fragments or variants thereof, or the region between the conserved cysteines of the IgV domain located at residues 63 and 170 of the full-length protein SEQ ID NO: 11, corresponding to the sequence set forth in SEQ ID NO: 102: CTYQMTSTPTQPIVIWKYKSFCRDRIADAFSPASVDNQLNAQLAAGNPGYNPYVE CQDSVRTVRVVATKQGNAVTLGDYYQGRRITITGNADLTFDQTAWGDSGVYYC

. In some further embodiments the LSR extracellular domain polypeptide consists

essentially of the amino acid of the IgV domain as set forth in any one of SEQ ID NO: 95, and SEQ ID NO: 102.

Generally, the LSR polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence of LSR can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. The signal peptide sequence that is used to replace the LSR signal peptide sequence can be any known in the art.

Optionally, the LSR ECD refers also to any one of the nucleic acid sequences encoding LSR ECD polypeptides, optionally to the nucleic acid sequences set forth in SEQ ID NO:40, 41, 132, 44, 155, 188, or fragments thereof and/or degenerative variants thereof, encoding LSR ECD polypeptides set forth in any one of SEQ ID NO: 12, 14, 47, 48, 49, 50, respectively.

Optionally, the LSR ECD refers to orthologous ECD polypeptides. Optionally, the LSR ECD refers to mouse LSR ECD polypeptides, set forth in SEQ ID NOs:21, and/or a mouse LSR ECD-IgG2a-Fc-fused polypeptide, set forth in SEQ ID NOs:26.

Variants of LY6G6F, VSIGIO, TMEM25 and/or LSR polypeptides

The present invention encompasses useful variants of LY6G6F, VSIGIO, TMEM25 and/or LSR polypeptides including those that increase biological activity, as indicated by any of the assays described herein, or that increase half life or stability of the protein. Soluble LY6G6F, VSIGIO, TMEM25 and/or LSR proteins or fragments, or fusions thereof having LY6G6F, VSIGIO, TMEM25 and/or LSR proteins activity, respectively, can be engineered to increase biological activity. In a further embodiment, the LY6G6F, VSIGIO, TMEM25 and/or LSR proteins or fusion protein is modified with at least one amino acid substitution, deletion, or insertion that increases the binding of the molecule to an immune cell, for example a T cell, and transmits an inhibitory signal into the T cell.

Other optional variants are those LY6G6F, VSIGIO, TMEM25 and/or LSR proteins that are engineered to selectively bind to one type of T cell versus other immune cells. For

example, the LY6G6F, VSIGIO, TMEM25 and/or LSR polypeptide can be engineered to bind optionally to Tregs, ThO, Thl, Thl7, Th2 or Th22 cells. Preferential binding refers to binding that is at least 10%, 20%, 30%, 40%, 50%, 60%f 70%, 80%, 90%, 95%, or greater for one type of cell over another type of cell. Still other variants of LY6G6F, VSIGIO, TMEM25 and/or LSR protein can be engineered to have reduced binding to immune cells relative to wildtype LY6G6F, VSIGIO, TMEM25 and/or LSR protein, respectively. These variants can be used in combination with variants having stronger binding properties to modulate the immune response with a moderate impact.

Also optionally, variant LY6G6F, VSIGIO, TMEM25 and/or LSR protein can be engineered to have an increased half-life relative to wildtype. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art.

The LY6G6F protein (SEQ ID NO: 1) also has the following non-silent SNPs

(Single Nucleotide Polymorphism) as listed in Table E, (given according to their position(s) on the amino acid sequence, with the alternative amino acid listed the presence of SNPs in LY6G6F protein (SEQ ID NO: l) sequence provides support for alternative sequence(s) of this protein according to the present invention. SEQ ID NO:58 is an example of such a alternative sequence, with alternative amino-acids, using part of the SNPs below

Table E - Amino acid mutations

SNP position(s) on amino Alternative amino acid(s)

acid sequence

34 P -> Q

39 P -> S

107 A -> T

167 R -> K

The LSR protein (SEQ ID NO: 11) also has the following non-silent SNPs (Single Nucleotide Polymorphism) as listed in Table F, (given according to their position(s) on the amino acid sequence, with the alternative amino acid listed; the presence of SNPs in LSR protein (SEQ ID NO: 11) sequence provides support for alternative sequence(s) of this protein according to the present invention. SEQ ID NO: 143 is an example of such a alternative sequence, with alternative amino-acids, using part of the SNPs below

Table F - Amino acid mutations

SN P position 1 ■> ) on ami 10 Λ II I'll ii 1 111 ino i K'i Ks>

acid scquona

209 I -> M

211 D -> G

260 L -> R

315 S -> N

382 A -> G

591 N -> D

The VSIG10 protein (SEQ ID NO:3) also has the following non-silent SNPs (Single Nucleotide Polymorphism) as listed in Table G, (given according to their position(s) on the amino acid sequence, with the alternative amino acid listed; the presence of SNPs in VSIG10 protein (SEQ ID NO: 3) sequence provides support for alternative sequence(s) of this protein according to the present invention.

Table G - Amino acid mutations

SNP position(s) on amino Alternative amino acid(s)

acid sequence

333 V -> M

435 H -> Y

The TMEM25 protein (SEQ ID NO:7) also has the following non-silent SNPs

(Single Nucleotide Polymorphism) as listed in Table H, (given according to their position(s) on the amino acid sequence, with the alternative amino acid listed; the presence of SNPs in TMEM25 protein (SEQ ID NO:7) sequence provides support for alternative sequence(s) of this protein according to the present invention.

Table H - Amino acid mutations

SNP position(s) on amino Alternative amino acid(s)

acid sequence

25 W -> C

342 Q -> R

Various aspects of the invention are described in further detail in the following subsections.

NUCLEIC ACIDS

A "nucleic acid fragment" or an "oligonucleotide" or a "polynucleotide" are used herein interchangeably to refer to a polymer of nucleic acid residues. A polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

Thus, the present invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 90%, at least 95, 96, 97, 98 or 99 % or more identical to the nucleic acid sequences set forth herein], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion. The present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention), which include sequence regions unique to the polynucleotides of the present invention.

Thus, the present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention. The present invention also encompasses homologues of these polypeptides, such homologues can be at least 90 %, at least 95, 96, 97, 98 or 99 % or more homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. As mentioned hereinabove, biomolecular sequences of the present invention can be efficiently utilized as tissue or pathological markers and as putative drugs or drug targets for treating or preventing a disease.

Oligonucleotides designed for carrying out the methods of the present invention for any of the sequences provided herein (designed as described above) can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.

The oligonucleotides of the present invention may comprise heterocyclic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.

Preferable oligonucleotides are those modified in either backbone, internucleoside linkages or bases, as is broadly described hereinunder. Such modifications can oftentimes facilitate oligonucleotide uptake and resistivity to intracellular conditions.

Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Patent Nos: 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821 ; 5,541,306; 5,550,111 ; 5,563,253; 5,571,799; 5,587,361 ; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141 ; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.

Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further bases include those disclosed in U.S. Pat. No: 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases are particularly useful for increasing the binding affinity of the

oligomeric compounds according to at least some embodiments of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

Another modification of the oligonucleotides according to at least some embodiments of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No: 6,303,374.

It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

PEPTIDES

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide" and "protein" include glycoproteins, as well as non-glycoproteins.

Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing.

In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3: 1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565.

It will be appreciated that peptides identified according to the teachings of the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=0, 0=C-NH, CH2-0, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.

Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (-CO-CH2-), OC-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide

derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxy lysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D-and L-amino acids.

Since the peptides of the present invention are preferably utilized in therapeutics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.

EXPRESSION SYSTEMS

To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element. As used herein, the phrase "cis acting regulatory element" refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.

Any suitable promoter sequence can be used by the nucleic acid construct of the present invention.

Preferably, the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1 :268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275] ; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). The nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.

The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., including Retro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred

constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining elements, or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptides of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

RECOMBINANT EXPRESSION VECTORS AND HOST CELLS

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a protein according to at least some embodiments of the invention, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Examples of vector types are plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". The invention is intended to include such forms of expression vectors, such as plasmids, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors according to at least some embodiments of the invention comprise a nucleic acid according to at least some embodiments of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors according to at least some embodiments of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors according to at least some embodiments of the invention can be designed for production of variant proteins in prokaryotic or eukaryotic cells. For example, proteins according to at least some embodiments of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, to the amino or C terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, PreScission, TEV and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector encoding for the protein of the invention is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, polypeptides of the present invention can be produced in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40. For other

suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

According to at least some embodiments the invention further provides a recombinant expression vector comprising a DNA molecule according to at least some embodiments of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to mRNA encoding for protein according to at least some embodiments of the invention. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.

According to at least some embodiments the invention pertains to host cells into which a recombinant expression vector according to at least some embodiments of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, protein according to at least some embodiments of the invention can be produced in bacterial cells such as E. coli, insect cells, yeast, plant or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS or 293 cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding protein according to at least some embodiments of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell according to at least some embodiments of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) protein according to at least some embodiments of the invention. Accordingly, the invention further provides methods for producing proteins according to at least some embodiments of the invention using the host cells according to at least some embodiments of the invention. In one embodiment, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector encoding protein according to at least some embodiments of the invention has been introduced) in a suitable medium such that the protein according to at least some embodiments of the invention is produced. In another embodiment, the method further comprises isolating protein according to at least some embodiments of the invention from the medium or the host cell.

For efficient production of the protein, it is preferable to place the nucleotide sequences encoding the protein according to at least some embodiments of the invention under the control of expression control sequences optimized for expression in a desired host. For example, the sequences may include optimized transcriptional and/or translational regulatory sequences (such as altered Kozak sequences).

It should be noted, that according to at least some embodiments of the present invention the LY6G6F, VSIG10, TMEM25 and/or LSR proteins according to at least some embodiments of the invention may be isolated as naturally-occurring polypeptides, or from any source whether natural, synthetic, semi-synthetic or recombinant. Accordingly, the LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be isolated as naturally-occurring proteins from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human. Alternatively, the LY6G6F, VSIG10, TMEM25 and/or LSR proteins may be isolated as recombinant polypeptides that are expressed in prokaryote or eukaryote host cells, or isolated as a chemically synthesized polypeptide.

A skilled artisan can readily employ standard isolation methods to obtain isolated LY6G6F, VSIGIO, TMEM25 and/or LSR proteins. The nature and degree of isolation will depend on the source and the intended use of the isolated molecules.

Transgenic Animals and Plants

According to at least some embodiments the invention also provides transgenic non-human animals and transgenic plants comprising one or more nucleic acid molecules according to at least some embodiments of the invention that may be used to produce the polypeptides according to at least some embodiments of the invention. The polypeptides can be produced in and recovered from tissue or bodily fluids, such as milk, blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.

Non-human transgenic animals and transgenic plants are produced by introducing one or more nucleic acid molecules according to at least some embodiments of the invention into the animal or plant by standard transgenic techniques. The transgenic cells used for making the transgenic animal can be embryonic stem cells, somatic cells or fertilized egg cells. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual 2ed. Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999).

GENE THERAPY

Accordiing to at least some embodiments of the present invention, nucleic acid sequences encoding soluble LY6G6F, VSIGIO, TMEM25 and/or LSR proteins can be used in gene therapy for treatment of infectious disorders, and/or immune related disorders, and or cancer.

As used herein, "gene therapy" is a process to treat a disease by genetic manipulation so that a sequence of nucleic acid is transferred into a cell, the cell then expressing any genetic product encoded by the nucleic acid. For example, as is well known by those skilled in the art, nucleic acid transfer may be performed by inserting an expression vector containing the nucleic acid of interest into cells ex vivo or in vitro by a variety of methods including, for example, calcium phosphate precipitation, diethyaminoethyl dextran, polyethylene glycol (PEG), electroporation, direct injection, lipofection or viral infection (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989); Kriegler M. Gene Transfer ad Expression: A Laboratory Manual (W. H. Freeman and Co, New York, N.Y., 1993) and Wu, Methods in Enzymology (Academic Press, New York, 1993). Alternatively, nucleic acid sequences of interest may be transferred into a cell in vivo in a variety of vectors and by a variety of methods including, for example, direct administration of the nucleic acid into a subject, or insertion of the nucleic acid into a viral vector and infection of the subject with the virus. Other methods used for in vivo transfer include encapsulation of the nucleic acid into liposomes, and direct transfer of the liposomes, or liposomes combined with a hemagglutinating Sendai virus, to a subject. The transfected or infected cells express the protein products encoded by the nucleic acid in order to ameliorate a disease or the symptoms of a disease.

ANTIBODIES AND IMMUNE SYSTEM RESPONSE

As used herein, the terms "immunologic", "immunological" or "immune" response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. Without wishing to be limited by a single hypothesis, a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

An "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

A "signal, transduction pathway" refers to the biochemical relationship between varieties of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.

As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell.

The term "antibody" as referred to herein includes whole polyclonal and monoclonal antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., LY6G6F, VSIG10, TMEM25 and/or LSR molecules, and/or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V Light, V Heavy, Constant light (CL) and CHI domains; (ii) a F(ab').2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds LY6G6F, VSIGIO, TMEM25 or LSR proteins and/or fragments thereof, and is substantially free of antibodies that specifically bind antigens other than LY6G6F, VSIGIO, TMEM25 or LSR, respectively. An isolated antibody that specifically binds LY6G6F, VSIGIO, TMEM25 or LSR proteins may, however, have cross-reactivity to other antigens, such as LY6G6F, VSIGIO, TMEM25 or LSR molecules from other species, respectively. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies according to at least some embodiments of the

invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or

transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a

recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

As used herein, an antibody that "specifically binds to human LY6G6F, VSIGIO, TMEM25 or LSR proteins" is intended to refer to an antibody that binds to LY6G6F, VSIGIO, TMEM25 or LSR proteins, respectively, such as for example, one with a KD of 5X10 -8 M, 3X10 -8 M, IX.10 -9 M or less.

The term "K-assoc" or "Ka", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdiss" or "Kd," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface Plasmon resonance, preferably using a biosensor system such as a BiacoreRTM. system.

As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD of 10-8 M or less, more preferably 10 -9 M or less and even more preferably 10 -10 M or less for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10 -7 M or less, more preferably 10 -8 M or less.

As used herein, the term "subject" or "patient" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

Anti-LY6G6F. anti-VSIGlO. anti-TMEM25 and anti-LSR Antibodies

The antibodies according to at least some embodiments of the invention including those having the particular germline sequences, homologous antibodies, antibodies with conservative modifications, engineered and modified antibodies are characterized by particular functional features or properties of the antibodies. For example, the antibodies bind specifically to human LY6G6F, VSIGIO, TMEM25 or LSR. Preferably, an antibody according to at least some embodiments of the invention binds to corresponding LY6G6F, VSIGIO, TMEM25 or LSR with high affinity, for example with a KD of 10 -8

M or less or 10 -9 M or less or even 10 -10 M or less. The anti-LY6G6F, anti- VSIGIO, anti-TMEM25 and anti-LSR antibodies according to at least some embodiments of the present invention preferably exhibit one or more of the following characteristics:

(i) binds to corresponding human LY6G6F, VSIGIO, TMEM25 or LSR with a KD of 5.X10 -8 M or less;

(ii) modulates (enhances or inhibits) B7 immune costimulation and related activities and functions such a T cell responses involved in antitumor immunity and autoimmunity, and/or

(iii) binds to LY6G6F, VSIGIO, TMEM25 or LSR antigen expressed by cancer cells including for example melanoma, cancers of liver, renal, brain, breast, colon, lung, ovary, pancreas, prostate, stomach, multiple myeloma, and hematopoietic cancer, including but not limited to lymphoma (Hodgkin's and non Hodgkin's), acute and chronic lymphoblastic leukemia and acute and chronic myeloid leukemia., but does not substantially bind to normal cells. In addition, preferably these antibodies and conjugates thereof will be effective in eliciting selective killing of such cancer cells and for modulating immune responses involved in autoimmunity and cancer.

More preferably, the antibody binds to corresponding human LY6G6F, VSIGIO, TMEM25 or LSR antigen with a KD of 3X10 -8 M or less, or with a KD of 1X10 -9 M or less, or with a KD of 0.1. X10 -9 M or less, or with a KD Of 0.05.X10 -9 M or less or with a KD of between 1X10 -9 and 1X10 -11 M.

Standard assays to evaluate the binding ability of the antibodies toward LY6G6F, VSIGIO, TMEM25 or LSR are known in the art, including for example, ELISAs, Western blots and RIAs. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore analysis.

Upon production of anti-LY6G6F, anti- VSIGIO, anti-TMEM25 and anti-LSR antibody sequences from antibodies can bind to LY6G6F, VSIGIO, TMEM25 or LSR the VH and VL sequences can be "mixed and matched" to create other anti-LY6G6F, anti-VSIG10, anti-TMEM25 and anti-LSR, binding molecules according to at least some embodiments of the invention. LY6G6F, VSIGIO, TMEM25 or LSR binding of such "mixed and matched" antibodies can be tested using the binding assays described above, e.g., ELISAs). Preferably, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH

sequence. Likewise, preferably a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence. For example, the VH and VL sequences of homologous antibodies are particularly amenable for mixing and matching.

ANTIBODIES HAVING PARTICULAR GERMLINE SEQUENCES

In certain embodiments, an antibody of the invention comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.

As used herein, a human antibody comprises heavy or light chain variable regions that is "the product of" or "derived from" a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody.

A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation. However, a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody may be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

HOMOLOGOUS ANTIBODIES

In yet another embodiment, an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to isolated anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR amino acid sequences of preferred anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies, respectively, wherein the antibodies retain the desired functional properties of the parent anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies.

As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available commercially), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules according to at least some embodiments of the invention. To obtain gapped alignments for comparison purposes,

Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDRl, CDR2 and CDR3 sequences and a light chain variable region comprising CDRl, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on preferred anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies isolated and produced using methods herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies according to at least some embodiments of the invention, respectively.

In various embodiments, the anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti- LSR antibody can be, for example, human antibodies, humanized antibodies or chimeric antibodies.

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody according to at least some embodiments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody according to at least some embodiments of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (j) above) using the functional assays described herein.

Antibodies that Bind to the Same Epitope as anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR according to at least some embodiments of the invention.

In another embodiment, the invention provides antibodies that bind to preferred epitopes on human LY6G6F, VSIG10, TMEM25 or LSR which possess desired functional properties such as modulation of B7 co-stimulation and related functions. Other antibodies with desired epitope specificity may be selected and will have the ability to cross-compete for binding to LY6G6F, VSIG10, TMEM25 or LSR antigen with the desired antibodies.

ENGINEERED AND MODIFIED ANTIBODIES

An antibody according to at least some embodiments of the invention further can be prepared using an antibody having one or more of the VH and/or VL sequences derived from an anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibody starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant regions, for example to alter the effector functions of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321 :522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A. 86: 10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)

Suitable framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet), as well as in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage" Eur. J Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

Another type of variable region modification is to mutate amino acid residues within the VH and/or VL CDR 1 , CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications (as discussed above) are introduced. The mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Engineered antibodies according to at least some embodiments of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

In addition or alternative to modifications made within the framework or CDR regions, antibodies according to at least some embodiments of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen- binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acid residues

329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. Nos. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acid positions

231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for Fc gamma RI, Fc gamma RII, Fc gammaRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII. Additionally, the following combination mutants are shown to improve Fcgamma.RIII binding: T256A/S298A, S298A E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and increase antibody circulation half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering

one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies according to at least some embodiments of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8.7- cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1 ,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that

antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-poly ethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies according to at least some embodiments of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

METHODS OF ENGINEERING ANTIBODIES

As discussed above, anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies having VH and VK sequences disclosed herein can be used to create new anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies, respectively, by modifying the VH and/or VL sequences, or the constant regions attached thereto. Thus, in another aspect according to at least some embodiments of the invention, the structural features of an anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibody according to at least some embodiments of the invention, are used to create structurally related anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR antibodies that retain at least one functional property of the antibodies according to at least some embodiments of the invention, such as binding to human LY6G6F, VSIG10, TMEM25 or LSR, respectively. For example, one or more CDR regions of one LY6G6F, VSIG10, TMEM25 or LSR antibody or mutations thereof, can be combined recombinantly with

known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-LY6G6F, anti- VSIGIO, anti-TMEM25 or anti-LSR antibodies according to at least some embodiments of the invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the VH and/or VK sequences provided herein, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VK sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequences is used as the starting material to create a "second generation" sequences derived from the original sequences and then the "second generation" sequences is prepared and expressed as a protein.

Standard molecular biology techniques can be used to prepare and express altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequences is one that retains one, some or all of the functional properties of the anti-LY6G6F, anti- VSIGIO, anti-TMEM25 or anti-LSR antibodies, respectively, produced by methods and with sequences provided herein, which functional properties include binding to LY6G6F, VSIGIO, TMEM25 or LSR antigen with a specific KD level or less and/or modulating B7 costimulation and/or selectively binding to desired target cells such as for example melanoma, cancers of liver, renal, brain, breast, colon, lung, ovary, pancreas, prostate, stomach, multiple myeloma and hematopoietic cancer, including but not limited to lymphoma (Hodgkin's and non Hodgkin's), acute and chronic lymphoblastic leukemia and acute and chronic myeloid leukemia, that express LY6G6F, VSIGIO, TMEM25 and/or LSR antigen.

The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein.

In certain embodiments of the methods of engineering antibodies according to at least some embodiments of the invention, mutations can be introduced randomly or selectively along all or part of an anti-LY6G6F, anti- VSIGIO, anti-TMEM25 or anti-LSR antibody coding sequence and the resulting modified anti-LY6G6F, anti- VSIGIO, anti- TMEM25 or anti-LSR antibodies can be screened for binding activity and/or other desired functional properties.

Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize physiochemical properties of antibodies.

NUCLEIC ACID MOLECULES ENCODING ANTIBODIES

Another aspect of the invention pertains to nucleic acid molecules that encode the antibodies according to at least some embodiments of the invention. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids according to at least some embodiments of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.

Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of anti-LY6G6F, anti-VSIGlO, anti-TMEM25 or anti-LSR Monoclonal Antibodies

Monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.

A preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g.,. human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to CabiUy et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

According to at least some embodiments of the invention, the antibodies are human monoclonal antibodies. Such human monoclonal antibodies directed against LY6G6F, VSIG10, TMEM25 and/or LSR can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as the HuMAb Mouse RTM and KM Mouse RTM, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb Mouse TM. (Medarex. Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy (.mu. and.gamma.) and.kappa. light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous.mu. and.kappa. chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or .kappa., and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGkappa. monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101 ; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of the HuMab Mouse RTM., and the genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; TuaiUon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; TuaiUon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591 ; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al.

In another embodiment, human antibodies according to at least some embodiments of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice TM.", are described in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti- LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSR antibodies according to at least some embodiments of the invention. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181 ; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-LY6G6F, anti- VSIGIO, anti-TMEM25 and/or anti-LSR antibodies according to at least some embodiments of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice" can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA 97:722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti- LY6G6F, anti- VSIGIO, anti-TMEM25 and/or anti-LSR antibodies according to at least some embodiments of the invention.

Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,73 1 ; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies according to at least some embodiments of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

IMMUNIZATION OF HUMAN IG MICE

When human Ig mice are used to raise human antibodies according to at least some embodiments of the invention, such mice can be immunized with a purified or enriched preparation of LY6G6F, VSIGIO, TMEM25 and/or LSR antigen and/or recombinant LY6G6F, VSIGIO, TMEM25 and/or LSR, or LY6G6F, VSIGIO, TMEM25 and/or LSR fusion protein, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 ; and PCT Publication WO 98/24884 and WO 01/14424. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, a purified or recombinant preparation (5-50.mu.g) of

LY6G6F, VSIG10, TMEM25 and/or LSR antigen can be used to immunize the human Ig mice intraperitoneally.

Prior experience with various antigens by others has shown that the transgenic mice respond when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. However, adjuvants other than Freund's are also found to be effective. In addition, whole cells in the absence of adjuvant are found to be highly immunogenic. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti- LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSR human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each immunization may need to be performed. Between 6 and 24 mice are typically immunized for each antigen. Usually both HCo7 and HCol2 strains are used. In addition, both HCo7 and HCol2 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCo 12). Alternatively or additionally, the KM Mouse. RTM. strain can be used.

GENERATION OF HYBRIDOMAS PRODUCING HUMAN MONOCLONAL ANTIBODIES

To generate hybridomas producing human monoclonal antibodies according to at least some embodiments of the invention, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 X 10 -5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After approximately two weeks, cells can be cultured in medium in

which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80 degrees C.

GENERATION OF TRANSFECTOMAS PRODUCING MONOCLONAL ANTIBODIES

Antibodies according to at least some embodiments according to at least some embodiments of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202).

For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segments within the vector and the VK segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors according to at least some embodiments of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or.beta.-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SR alpha, promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors according to at least some embodiments of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies according to at least some embodiments of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are

produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

CHARACTERIZATION OF ANTIBODY BINDING TO ANTIGEN

Antibodies according to at least some embodiments of the invention can be tested for binding to LY6G6F, VSIG10, TMEM25 and/or LSR by, for example, standard ELISA. Briefly, microtiter plates are coated with purified LY6G6F, VSIG10, TMEM25 and/or LSR at 0.25.mu.g/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from -immunized mice) are added to each well and incubated for 1-2 hours at 37 degrees C. The plates are washed with PBS/Tween and then incubated with secondary reagent (e.g., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37 degrees C. After washing, the plates are developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen for hybridomas that show positive reactivity with LY6G6F, VSIG10, TMEM25 and/or LSR immunogen. Hybridomas that bind with high avidity to LY6G6F, VSIG10, TMEM25 and/or LSR are subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140 degrees C., and for antibody purification.

To purify anti-LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSR antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80 degrees C.

To determine if the selected anti-LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSRmonoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, 111.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using LY6G6F, VSIG10, TMEM25 and/or LSR coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of microtiter plates can be coated with l.mu.g/ml of anti-human immunoglobulin overnight at 4 degrees C. After blocking with 1% BSA, the plates are reacted with lmug /ml or less of test monoclonal antibodies or purified isotype controls, at ambient temperature for one to two hours. The wells can then be reacted with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.

Anti-LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSRhuman IgGs can be further tested for reactivity with LY6G6F, VSIG10, TMEM25 and/or LSR antigen, respectively, by Western blotting. Briefly, LY6G6F, VSIG10, TMEM25 and/or LSRantigen can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

ALTERNATIVE SCAFFOLDS

According to at least some embodiments the invention relates to protein scaffolds with specificities and affinities in a range similar to specific antibodies. According to at least some embodiments the present invention relates to an antigen-binding construct comprising a protein scaffold which is linked to one or more epitope-binding domains. Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques. According to at least some embodiments the invention relates to alternative scaffolds including, but not limited to, anticalins, DARPins, Armadillo repeat proteins, protein A, lipocalins, fibronectin domain, ankyrin consensus repeat domain, thioredoxin, chemically constrained peptides and the like. According to at least some embodiments the invention relates to alternative scaffolds that are used as therapeutic agents for treatment of cancer, autoimmune and infectious diseases as well as for in vivo diagnostics.

According to at least some embodiments the invention further provides a pharmaceutical composition comprising an antigen binding construct as described herein a pharmaceutically acceptable carrier.

The term 'Protein Scaffold' as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions. Such protein scaffolds may comprise antigen- binding sites in addition to the one or more constant regions, for example where the protein scaffold comprises a full IgG. Such protein scaffolds will be capable of being linked to other protein domains, for example protein domains which have antigen- binding sites, for example epitope-binding domains or ScFv domains.

A "domain" is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. A "single antibody variable domain" is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.

The phrase "immunoglobulin single variable domain" refers to an antibody variable domain (VH , V HH, V L) that specifically binds an antigen or epitope independently of a different V region or domain. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other, different variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the

immunoglobulin single variable domain binds antigen independently of the additional variable domains). A "domain antibody" or "dAb" is the same as an "immunoglobulin single variable domain" which is capable of binding to an antigen as the term is used herein. An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V HH dAbs. Camelid V HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such V HH domains may be humanised according to standard techniques available in the art, and such domains are still considered to be "domain antibodies" according to the invention. As used herein "VH includes camelid V HH domains. NARV are another type of immunoglobulin single variable domain which were identified in cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen Receptor variable region (commonly abbreviated to V(NAR) or NARV). For further details see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.

The term "epitope-binding domain" refers to a domain that specifically binds an antigen or epitope independently of a different V region or domain, this may be a domain antibody (dAb), for example a human, camelid or shark immunoglobulin single variable domain or itmay be a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans- body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human γ-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; Armadillo repeat proteins, thioredoxin, and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a ligand other than the natural ligand.

Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties i.e. Evibodies. For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001 ) Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid secondary structure with a numer of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B 1 and US20070224633. An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomisation of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1 Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007) A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two alpha helices;-beta turn. They can be engineered to bind different target antigens by randomising residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. MoT Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. MoT Biol. 369, 1015-1028 (2007) and US20040132028 Al.

Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta;-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel. 18, 435- 444 (2005), US200801 39791, WO2005056764 and US6818418B 1.

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained

variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther.

5. 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and cono toxin and knottins. The microproteins have a loop which can be engineered to include upto 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see WO2008098796.

Other epitope binding domains include proteins which have been used as a scaffold to engineer different target antigen binding properties include human γbeta-crystallin and human ubiquitin (affilins), kunitz type domains of human protease inhibitors, PDZ- domains of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7 - Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15: 14-27 (2006). Epitope binding domains of the present invention could be derived from any of these alternative protein domains.

CONJUGATES OR IMMUNO CONJUGATES

The present invention encompasses conjugates for use in immune therapy comprising the LY6G6F, VSIG10, TMEM25 and/or LSR antigen and soluble portions thereof including the ectodomain or portions or variants thereof. For example the invention encompasses conjugates wherein the ECD of the LY6G6F, VSIG10, TMEM25 and/or LSR antigen is attached to an immunoglobulin or fragment thereof. The invention contemplates the use thereof for promoting or inhibiting LY6G6F, VSIG10, TMEM25 and/or LSR antigen activities such as immune costimulation and the use thereof in treating transplant, autoimmune, and cancer indications described herein.

In another aspect, the present invention features antibody-drug conjugates (ADCs), used for example for treatment of cancer, consisting of an antibody (or antibody fragment such as a single-chain variable fragment [scFv]) linked to a payload drug (often cytotoxic). The antibody causes the ADC to bind to the target cancer cells. Often the ADC is then internalized by the cell and the drug is released into the cell. Because of the targeting, the side effects are lower and give a wider therapeutic window. Hydrophilic linkers (e.g., PEG4Mal) help prevent the drug being pumped out of resistant cancer cells through MDR (multiple drug resistance) transporters. ADCs based on

cleavable linkers are thought to have a less favorable therapeutic window, but targets (tumor cell surface antigens) that do not get internalized efficiently seem more suitable for cleavable linkers.

In another aspect, the present invention features immunoconjugates comprising an anti-LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSR antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to an antibody according to at least some embodiments of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg.TM.; Wyeth).

Cytotoxins can be conjugated to antibodies according to at least some embodiments of the invention using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P. A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091 ; Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131, indium 111, yttrium 90 and lutetium 177. Methods for preparing radio immunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin (IDEC Pharmaceuticals) and Bexxar. (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies according to at least some embodiments of the invention.

The antibody conjugates according to at least some embodiments of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-. gamma.; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The

Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

BISPECIFIC MOLECULES

In another aspect, the present invention features bispecific molecules comprising an anti-LY6G6F, anti- VSIGIO, anti-TMEM25 and/or anti-LSR antibody, or a fragment thereof, according to at least some embodiments of the invention. An antibody according to at least some embodiments of the invention, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody according to at least some embodiments of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule according to at least some embodiments of the invention, an antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for LY6G6F, VSIGIO, TMEM25 and/or LSRand a second binding specificity for a second target epitope. According to at least some embodiments of the invention, the second target epitope is an Fc receptor, e.g., human Fc gamma RI (CD64) or a human Fc alpha receptor (CD89). Therefore, the invention includes bispecific molecules capable of binding both to Fc gamma. R, Fc alpha R or Fc epsilon R expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing LY6G6F, VSIGIO, TMEM25 and/or LSR, respectively. These bispecific molecules target LY6G6F, VSIGIO, TMEM25 and/or LSR expressing cells to effector cell and trigger Fc receptor-mediated effector cell activities, such as phagocytosis of an LY6G6F, VSIGIO, TMEM25 and/or LSRexpressing cells,

antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

According to at least some embodiments of the invention in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-6f binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell.

The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhan
This document is too large, please check out its pdf version from the Documents tab!