ANTI-LAMP1 ANTIBODIES AND ANTIBODY DRUG CONJUGATES, AND USES

THEREOF

Antibodies are provided which specifically bind human and Macaca fascicularis lysosomal-associated membrane protein 1 (LAMP1 ) proteins and immunoconjugates comprising said antibodies conjugated or linked to a growth inhibitory agent. Pharmaceutical compositions comprising antibodies or immunoconjugates of the invention and use of the antibodies or immunoconjugates for the treatment of cancer are also provided, as well as LAMP1 antibodies, isolated nucleic acids, vectors and host cells comprising a sequence encoding said antibodies and the use of said antibody as a diagnostic tool. The application further provides for the detection of LAMP1 gene amplification or gain in cancer cells leading to the determination if patients with cancer are likely to respond to anti-LAMP1 therapy. Therefore, it is proposed an in vitro method of selecting patients with cancer which comprises determining, in a biological sample of a patient with cancer which includes cancer cells, if said patient harbors a LAMP1 gene copy number gain; and selecting the patient based on the presence of LAMP1 gene copy number gain. Anti-LAMP1 therapeutic agent for use for treating cancer in a patient harboring LAMP1 gene copy number gain in cancer cells is further provided.

Lysosome-associated membrane protein 1 (LAMP1 ), also known as CD107 antigen-like family member A (CD107a), is a single-pass type I membrane protein, which belongs to the LAMP family. LAMP2 is the closest member of the family and both proteins are the most abundant glycoproteins within the lysosomal membrane (Sawada, R. et al., 1993, J Biol Chem 268: 12675-12681 ).

Although encoded by separate genes, with LAMP1 located on chromosome 13q34 and LAMP2 on Xq24-25, the proteins are similar in their primary structure, with -36% sequence homology (Mattei, M.G. et al., 1990, J Biol Chem 265:7548-7551 ). LAMP1 and LAMP2 consist of a polypeptide core of approximately 40kDa; they are both anchored via their C-terminus to the lysosomal membrane and expose the largest part, extensively glycosylated, to the lumenal side of lysosomes. Both proteins are among the most heavily glycosylated of cellular proteins with -50% of their mass as carbohydrates and these glycosylations seem to be the key for maintaining lysosome acidity and protecting the lysosomal membranes from autodigestion. However, the full biological function of these two highly glycosylated proteins in particular LAMP1 still needs to be elucidated (Fukuda, M., 1991 , J Biol Chem, 266:21327-21330; Winchester, B., 2001 ,

European Journal of Paediatry Neurology, 5:1 1-19; Gasnier, B., 2009 Biochimica et Biophysica Acta 1793:636-649).

LAMP1 is highly expressed in late endosomes and lysosomes designating LAMP1 as marker for these two organelles (Cook, N.R. et al., 2004, Traffic, 5 (9): 685-699). Thus, most of the literature on LAMP1 relates to endocytosis, pinoscyosis, or phagocytosis (Cook, N.R. et al., 2004, Traffic, 5 (9): 685-699).

Although the majority of LAMP1 and LAMP2 reside in the lysosome, some LAMP1 and LAMP2 immunoreactivity is also observed at low levels at the plasma membrane. The LAMP1 found in the plasma membrane represents only 1 -2% of total LAMP1. This is for example true for peripheral blood lymphocytes (Holcombe, R.F. et al. 1993, Clin Immunol Immunopathol. 67(1 ): 31 -39) and platelets (Silverstein, R.L. and Febbraio, M., 1992, Blood 80: 1470-1475).

Increased cell surface expression of LAMP1 and LAMP2 has been observed in tumor cell lines, for example in highly metastatic colonic carcinoma L4 cells (Saitoh, O. et al., 1992, J Biol Chem 267: 5700-571 1 ), on human metastasizing melanoma A2058, HT1080 (human fibrosarcoma), CaCo-2 (human colon-adenocarcinoma) cells and in colorectal neoplasms (Furuta, K. et al., 2001 , J Pathol 159 (2): 449-455).

The chromosomal region 13q34 in which LAMP1 is located has recently been linked to amplification events including a larger amplicon that involves CUL4A, LAMP1, TFDP1, and GAS6 in human breast cancer (Abba, Martin C. et al.; Cancer Res 2007; 4104). TFDP1 and perhaps CUL4A were identified in the above mentioned publication as the leading genes driving the amplification phenomenon. In particular, analysis of publicly available breast cancer gene expression (microarrays) data sets indicated that TFDP1 overexpression is associated with estrogen receptor (ER)-negative and high-grade breast carcinomas, as well as shorter overall survival, relapse-free survival, and metastasis-free interval. Conversely, LAMP1 expression did not significantly correlate with tumor grade. In the end, Abba et al. did not report that LAMP1 amplification translated into LAMP1 overexpression in human breast cancer cells.

The 1 1 amino-acid cytoplasmic tail of LAMP1 contains a 7 amino-acid linker sequence and a 4 amino acid long tyrosine motif (YQTI). It was shown that small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes. Mutations within said tyrosine motif were shown to have an impact on the binding of LAMP1 to adaptor proteins leading as well to impaired sorting (Obermijiler, S. et al., 2002, Journal of Cell Science 1 15: 185-194; Rohrer, J. et a/., 1996, Journal of Cell Biology 132(4): 565-576). Therefore, the abnormal cell surface expression of LAMP1 in different cancer cell lines might be related to mutations in the cytoplasmic tail even though the mechanism is still unclear. Furthermore, it has been shown that certain point mutations in the cytoplasmic tail lead to plasma membrane accumulation (Gough, N.R. et al., 1999, Journal of Cell Science 1 12 (23): 4257-4269).

Due to the fact that LAMP1 is a marker for endosomes and lysosomes, numerous commercially available anti-LAMP1 antibodies were developed for research purposes. These antibodies are either polyclonal or monoclonal and are restricted to some biochemical application such as immunohistochemistry (IHC), Western blots (WB), Fluorescence activated cell sorter (FACS) analysis, Immunoprecipitation (IP) and Enzyme-linked immunosorbent assay (ELISA).

LAMP1 protein also has been detected at the cell membrane of tumor cells.

E. Venetsanakos (WO 2005/012912) suggested that LAMP1 is expressed on the surface of colon cancer cells but not on the surface of normal colon cells and proposed that tumor growth might be reduced by targeting a cytotoxic agent to LAMP1 via an anti-LAMP1 antibody. Venetsanakos did not describe, however, preparation of anti-LAMP1 antibodies or conjugates thereof with cytotoxic or cytostatic agent or any data supporting his hypothesis. Indeed, though a decade has passed since Venetsanakos' initial filing and no anti-LAMP1 antibodies or their use as immunoconjugates in an anti-LAMP1 therapy has entered clinical development, so far. Accordingly, a great need exists for anti-LAMP1 antibodies or immunoconjugates for the treatment of cancer.

Definitions

As used herein "LAMP1 " designates the "Lysosomal associated membrane protein 1 ", a member of a family of glycoproteins that is also known as LAMPA, CD107a or LGP120. LAMP1 is, according to protein expression data for human tumoral samples in comparison to non tumoral samples presented in the following Example 5, expressed at the cell surface of colon adenocarcinomas, gastrointestinal tumors (small intestine, rectum, parotid gland), vital organs tumors (lung, liver, stomach, pancreas and kidney), reproductive organ tumors (breast, ovary and prostate) as well as skin, larynx and soft tissue tumors.

The human gene LAMP1 is found on chromosome 13q34 (1 13,951 ,469 -1 13,977,441 ) and has a total length of 26,273 kb.

A reference sequence of the cDNA coding for full-length human LAMP1 , including

the sequence encoding the signal peptide, is available from the GenBank database under accession number NM_005561 .3 (SEQ ID NO: 23) and the representative protein sequence, including the signal peptide (positions 1 -28) is available under NP_005552.3 (SEQ ID NO: 24). One potential isoform of LAMP1 has been reported which would miss the amino acids at positions 136-188 of SEQ I D NO: 24, corresponding to exon 4 of the gene coding for human LAMP1 . No synonymous SNPs have been identified in Caucasian population of at least 60 individuals.

Concerning its orthologs, human LAMP1 shares 66% sequence identity with respectively mouse LAMP1 (NP_034814, SEQ ID NO: 25) and rat LAMP1 (NP_036989, SEQ ID NO: 26), and human and Macaca mulatta LAMP1 (XP_001087801 , SEQ ID NO: 27) share 96% sequence identity.

The sequence of LAMP1 from Macaca mulatta (SEQ ID NO: 27) and the predicted sequence of Macaca fascicularis (SEQ ID NO: 39) are identical to 99%, said sequences differing by one additional leucine at position 1 1 of Macaca mulatta LAMP1 (SEQ ID NO: 27), i.e. in the signal peptide. Accordingly the sequences of mature LAMP1 from Macaca mulatta and Macaca fascicularis are identical.

The closest member of the LAMP family is LAMP 2 (P13473, human LAMP2, soluble LAMP2 protein SEQ ID NO: 40). Human LAMP1 and LAMP2 proteins share -36% sequence identity, and comprise some conserved glycosylation sites.

A "domain" may be any region of a protein, generally defined on the basis of sequence homologies and often related to a specific structural or functional entity. The domain organization of LAMP1 has not been entirely published so far.

Human LAMP1 consists of 417 amino acid residues and 28 amino-terminal residues corresponding to a cleavable signal peptide. The major portion of LAMP1 resides on the lumenal side of the lysosome and is heavily glycosylated by N-glycans. LAMP1 contains 18 potential N-glycosylation sites of which 5 are occupied with poly-N-acetyllactosamine glycans (Carlsson, S.R. and Fukuda, M., 1990, J. Biol. Chem. 265(33): 20488-20495). They are clustered into two domains separated by a hinge-like structure enriched with prolines and serines many being linked to O-glycans. LAMP1 has one transmembrane domain consisting of 24 hydrophobic amino acids near the COOH terminus, and contains a short cytoplasmic segment composed of 1 1 amino acid residues at the COOH-terminal end.

The nomenclature of the two domains of LAMP1 , "the first lumenal domain" and the "second lumenal domain" are based on the orientation of LAMP1 within its original

localization, the lysosome. Nevertheless, when LAMP1 is expressed at the cell surface, the two lumenal domains become extracellular domains, and therefore exposed at the cell surface. Therefore, in one embodiment "extracellular" in context of the invention refers to LAMP1 protein constructs comprising the first and/or second luminal domain(s) of LAMP1 as defined below and/or variants thereof.The domain organisation of human LAMP1 according to NP_005552.3 (SEQ ID NO: 24) has been mapped in example 6.1 and will be used in this document as follows:

Table 1 : Description of human LAMP1 domains

Accordingly, the domain consisting of the first to third loops of human LAMP1 consists of amino acids at positions 29-309 of SEQ ID NO: 24.

Domain organisation of Macaca fascicularis LAMP1 according to the predicted sequence (SEQ ID NO: 39) is as follows:

Table 2: Description of Macaca fascicularis LAMP1 domains

Accordingly, the domain consisting of first to third loops of Macaca fascicularis LAMP1 consists of amino acids at positions 27-307 of SEQ ID NO: 39.

A sequence alignment of human and Macaca fascicularis LAMP1 full-length proteins is shown on Figure 1.

The loop region 4 of human and Macaca fascicularis LAMP1 do not contain any glycosylation site, which distinguishes Loop 4 from Loops 1 -3 of LAMP1.

Loops 1 -4 have been defined from the primary amino acid sequence, and has been mapped in example 6.1 , but not from the 3D structure of LAMP1 since the structure was not solved prior to this work.

A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. A region encoding an expression product present in the DNA is called "coding DNA sequence" or "CDS".

As used herein, references to specific proteins (e.g., antibodies) can include a polypeptide having a native amino acid sequence, as well as variants and modified forms regardless of their origin or mode of preparation. A protein which has a native amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such native sequence proteins can be isolated from nature or can be prepared using standard recombinant and/or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternatively spliced forms), naturally occurring allelic variants and forms including post-translational modifications. A native sequence protein includes proteins following post-translational modifications such as glycosylation, or phosphorylation, or other modifications of some amino acid residues.

As used herein, the term "marker" refers to any biological, chemical or physical mean allowing identifying the presence, and possibly quantifying the expression of a target gene and/or protein in a biological sample. Such markers are well known from one skilled in the art. Advantageously, the markers according to the invention are genetic markers and/or protein markers.

The term "gene" means a DNA sequence that codes for, or corresponds to, a

particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

As used herein, the terms "copy number variation", "copy number variant" and "CNV" are used indifferently and refer to a DNA segment of 1 kb or larger and present at variable copy number in comparison with a reference genome. The terms "structural variant", "duplicon", "indel", "intermediate-sized structural variant (ISV)", "low copy repeat (LCR)", "multisite variant (MSV)", "paralogous sequence variant (PSV)", "segmental duplication", "interchromosomal duplication", and "intrachromosomal duplication", found in the literature, are included herein in the term "CNV".

Furthermore, copy number variation can refer to a single gene, or include a contiguous set of genes.

As used herein "gene number" describes the numbers of genes present in the cell. In diploid organisms, in a normal state, two copies of each nucleic sequence are naturally present in the genome, therefore, the copy number (CN) is =2. In particular, the genome displays two alleles for each gene, one on each chromosome of a pair of homologous chromosomes (except for the genes localized on sexual chromosomes).

Herein the word "gene number" and "gene copy number" can be used interchangeably.

In the context of the invention, a "copy" of a sequence encompasses a sequence identical to said sequence but also allelic variations of said sequence.

One example to measure DNA copy number and therefore DNA copy number change is array-based CGH which is a high-throughput technique to measure DNA copy number change across the genome. The DNA fragments or "clones" of test and reference samples are hybridized to mapped array fragments. Log2 intensity ratios of test to reference provide useful information about genome-wide profiles in copy number.

The "Log2" or "Log2 ratio" value is used to describe the copy number of a gene or a DNA fragment in a cell genome. In an ideal situation, the log2 ratio of normal (copy-variation neutral) clones is log2(2/2) = 0, single copy losses is log2(1/2) = -1 , and single copy gains is log2(3/2) = 0.58. Multiple copy gains or amplifications would have values of log2(4/2), log2(5/2),....

As used herein, the term "gain" of a sequence refers in general to the presence of a copy number≥ 2.5 (alternatively a Log2ratio≥ 0.32) of said sequence in the diploid genome of a subject. These≥ 2.5 copies may be adjacent or not on the genome; in particular they may be present in different regions of a pair of chromosomes or on chromosomes belonging to distinct pairs of chromosomes of the genome.

Accordingly, the term "gene copy number gain" refers to the presence of ≥2.5 copy numbers (alternatively a Log2ratio≥ 0.32) of a specific gene in the diploid genome of a subject. When the copy number =2.5, 50% of the cells used for defining the copy number contain the usual 2 copies of the gene in a diploid organism and 50% of the cells used for defining the copy number contain the usual 2 copies and 1 additional copy more of said gene (in total 3 copies of said gene).

The term "low gain" of a sequence refers in general to the presence of a copy number≥ 2.5 but <5 (alternatively 0.32< log2 ratio <1 .32) of said sequence in the diploid genome of a subject. The terms "amplification", "Amp", or "high gain" refer herein to the presence of a copy number≥5, or alternatively a Log2≥ 1.32, of a specific sequence in the diploid genome of a subject. Accordingly, the term "gene number amplification" refers to the presence of ≥ 5 copy numbers of a specific gene in the diploid genome of a subject

As used herein, a "fragment of a sequence" corresponds to a portion of said sequence, for instance of a nucleotide sequence. Said fragment is preferably at least 10 bp long. More preferably said fragment is at least 15 bp long, in particular at least 20 bp long. Most preferably, said fragment is at least 25 bp long, at least 30 bp long, in particular at least 33 bp long. A fragment of the above sequence may be in particular a primer or probe.

In the context of the invention, a "mutated sequence" of a reference sequence refers to a sequence including insertion(s), deletion(s) or substitution(s) of one or more nucleotide(s), wherein said mutated sequence is at least 75% identical to the reference sequence. The percentage of sequence identity is calculated by comparing the mutated sequence optimally aligned with the reference sequence, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions of the reference sequence, and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the mutated sequence is at least 80%, 85%, 90%, 95% identical to the reference sequence.

Preferably said mutated sequence of a reference sequence is an allelic variant of said reference sequence. As used herein, an "allelic variant" denotes any of two or more alternative forms of a gene occupying the same chromosome locus.

A sequence "at least 85% identical to a reference sequence" is a sequence having, on its entire length, 85%, or more, in particular 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence.

A percentage of "sequence identity" may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison is conducted by global pairwise alignment, e.g. using the algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970). The percentage of sequence identity can be readily determined for instance using the program Needle, with the BLOSUM62 matrix, and the following parameters gap-open=10, gap-extend=0.5.

A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophane, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

"Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively.

As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

In the context of the invention, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc, M.P. et al., 2003, Dev Comp Immunol. 27(1 ): 55-77; www.imgt.org).

As used herein, the term "antibody" denotes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanised, bispecific or multispecific antibodies.

As used herein, antibody or immunoglobulin also includes "single domain antibodies" which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit and bovine. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains. In particular, Camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain.

The variable heavy chain of these single domain antibodies devoid of light chains are known in the art as "VHH" or "nanobodv". Similar to conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional nanobodies can be produced by in vitro expression while achieving high yield. Furthermore, nanobodies are very stable, and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov; 77(1 ): 13-22).

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.

The term "chimeric antibody" refers to an engineered antibody which in its broadest sense contains one or more regions from one antibody and one or more regions from on or more other antibody(ies). In particular a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens. In an embodiment, a chimeric antibody has variable domains of mouse origin and constant domains of human origin

The term "humanised antibody" refers to an antibody which is initially wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. In an embodiment, a humanized antibody has constant domains of human origin.

"Fragments" of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

The term "Fab" denotes an antibody fragment having a molecular weight of about

50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. "dsFv" is a VH::VL

heterodimer stabilised by a disulphide bond. "(dsFv)2" denotes two dsFv coupled by a peptide linker.

The term "bispecific antibody" or "BsAb" denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1 .

The term "multispecific antibody" denotes an antibody which combines the antigen-binding sites of two or more antibodies within a single molecule.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

The term "hybridoma" denotes a cell, which is obtained by subjecting a B cell prepared by immunizing a non-human mammal with an antigen to cell fusion with a myeloma cell derived from a mouse or the like which produces a desired monoclonal antibody having an antigen specificity.

By "purified" and "isolated" it is meant, when referring to a polypeptide (i.e. the antibody of the invention) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of biological macromolecules of the same type are present. An "isolated" nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. In particular a subject according to the invention is a human.

Throughout the instant application, the term "comprising" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment

wherein no features other than the specifically mentioned features are present (i.e. "consisting of").

Throughout the instant application, the term "and/or" is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. For example, the sentence "quantifying the expression of a target gene and/or protein in a biological sample" indicates the expression of a target gene may be quantified (mRNA), or the expression of a protein or the expression of a target gene (mRNA) and the protein together may be quantified.

Accordingly, the wording "a variable domain of heavy chain of sequence SEQ ID NO: 1 or a sequence at least 85% identical thereto and/or a variable domain of light chain of sequence of sequence SEQ ID NO: 5, or a sequence at least 85% identical thereto" is to b interpreted as "a variable domain of heavy chain of sequence SEQ ID NO: 1 or a sequence at least 85% identical thereto" or "a variable domain of light chain of sequence of sequence SEQ ID NO: 5, or a sequence at least 85% identical thereto" or "a variable domain of heavy chain of sequence SEQ ID NO: 1 or a sequence at least 85% identical thereto and a variable domain of light chain of sequence of sequence SEQ ID NO: 5, or a sequence at least 85% identical thereto".

The term "cancer", "neoplasm", "tumor", and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively autonomous growth, so that they can exhibit an aberrant growth phenotype characterized by significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include precancerous (e.g. benign), malignant, metastatic, and non-metastatic cells.

Immunoconjugates

For therapeutic purposes, it is advantageous to create an antibody with optimal characteristics for use as an antibody drug conjugate, i.e. an antibody which specifically recognizes a target present on the surface of cancer cells and which is capable of efficiently triggering internalization once bound to said target.

The inventors raised antibodies against colon tumor cells or lung tumor cells and screened resulting clones for the differential binding to tumor cells and non-tumor tissue.

The inventors identified in this way antibodies distinguishing tumoral from non-tumoral tissues. Three of those antibodies were selected (the so-called antibodies "MAb1 ", "MAb2" and "MAb3"), fulfilling the expected features necessary for therapeutical application, in particular in the form of ADC. Those three antibodies showed high binding affinity (within the nanomolar range) to cell surface expressed LAMP1 in cancer cells. Furthermore, those three anti-LAMP1 antibodies showed high capacity to trigger internalization of the LAMP1/anti-LAMP1 antibody complex, as shown in example 4.4 and 4.3.

The inventors demonstrated that the chimeric antibodies derived from MAb1 , MAb2, MAb3 (chMAbl , chMAb2, chMAb3), combined with a cytotoxic maytansinoid (DM4) showed as well a high but slightly different binding affinity to human LAMP1 or cynomologus monkey LAMP1 then the naked antibody as shown in example 8.1 .7 and 8.1.8.

Accordingly in one embodiment the immunoconjugate in context of the invention has an affinity (EC50) for full length human LAMP1 and cynomologues monkey LAMP1 expressed at the cell surface of a recombinant cell line, wherein the cell line may be HCT1 16 and the apparent affinity measured via Flow Cytometry is < 30nM, for example <20nM or < 15nM.

The Methods to measure the affinity (EC50) for full length human LAMP1 and cynomologues monkey LAMP1 are further explained in the chapter "antibodies".

The inventors additionally demonstrated that a chimeric antibody derived from MAb1 (chMAbl ), combined with a cytotoxic maytansinoid (DM4), induces cytotoxic activity in vitro on human HCT1 16 tumor cells containing a stable integration of the LAMP1 coding DNA sequence in the genomic DNA and expressing LAMP1 on their surface.

Furthermore, the inventors demonstrated that humanized antibodies derived from MAb1 (huMAb1_1 , huMAb1_2, huMAb1_3), combined with a cytotoxic maytansinoid (DM4) induce cytotoxic activity in vitro on human HCT1 16 tumor cells containing a stable integration of the LAMP1 coding DNA sequence in the genomic DNA.

They have also shown that the immunoconjugate DM4-SPDB-chMAb1 induces a marked anti-tumor activity in vivo in mice bearing the primary human colon adenocarcinoma xenograft derived from patient CR-LRB-010P, when used at a dose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg, with a single injection, as described in example 10.1 .1 .

Furthermore, the inventors showed that this immunoconjugate induces a marked anti-tumor activity in vivo in mice bearing the primary human lung tumor xenograft derived from patient LUN-NIC-0014, when used at a dose of 10 mg/kg, 5 mg/kg and 2.5

mg/kg, with a single injection, as described in example 10.1 .2.

They have also shown that the immunoconjugates DM4-SPDB-huMAb1_1 , DM4-SPDB-chMAb2, and DM4-SPDB-chMAb3 induce a marked anti-tumor activity in vivo in different patient-derived xenograft as shown in example 10.2-10.4.

For example, it was shown the immunoconjugate DM4-SPDB-huMAb1_1 induces a marked anti-tumor activity in vivo in a primary human invasive ductal carcinoma xenograft and primary human lung tumor xenograft derived from patient, when used at a dose of 10 mg/kg, 5 mg/kg, 2.5 mg/kg, or 1.25 mg/kg with a single injection, as described in example 10.2.2 and 10.2.3.

Also the immunoconjugates DM4-SPDB-chMAb2 and DM4-SPDB-chMAb3 induced a marked anti-tumor activity in vivo in a murine model of primary human invasive ductal carcinoma xenograft derived from patient, when used at a dose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg or 5 mg/kg, 2.5 mg/kg and 1 .25 mg/kg, respectively, with a single injection, as described in example 10.3.2 and 10.4.

Altogether, for the first time, these results validly identify LAMP1 as a therapeutic target for the treatment of cancer.

Accordingly, the invention relates to an immunoconjugate comprising an antibody which:

a) binds to human and Macaca fascicularis LAMP1 proteins; and

b) is linked or conjugated to at least one growth inhibitory agent.

Any antibody which binds to human and Macaca fascicularis LAMP1 proteins, as described throughout the instant application (e.g. MAb4, fragments thereof, or chimeric or humanised version thereof), can be incorporated in the immunoconjugate according to the invention.

As used herein, "conjugate", "immunoconjugate", "antibody-drug conjugate" or

"ADC" have the same meaning and are interchangeable.

A "growth inhibitory agent", or "anti-proliferative agent", which can be used indifferently, refers to a compound or composition which inhibits growth of a cell, especially tumour cell, either in vitro or in vivo. A growth inhibitory agent denotes in particular a cytotoxic agent or a radioactive isotope.

The term "radioactive isotope" is intended to include radioactive isotopes suitable for treating cancer, such as At211, Ac225, Bi212, Bi213, Pb212, Er169, I131, I124, I125, Y90, In111, P32, Re186, Re188, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu such as Lu177. Such radioisotopes generally emit mainly beta-radiation. In an embodiment the

radioactive isotope is alpha-emitter isotope, more precisely Thorium 227 (Th227) which emits alpha-radiation. The immunoconjugates according to the present invention can be prepared as described in the application WO2004/091668.

In one embodiment, a radioactive isotope is selected from the group consisting of At211, Ac225, Bi213, Pb212, Er169, I124, I125, In111, P32, Re186, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu, for instance from At211, Er169, I125, In111, P32, Re186, Sm153, Sr89, radioactive isotopes of Lu, and Th227.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a drug or a pro-drug of a compound consisting in an anti-tubulin agent such as taxoids or taxanes, a vinca-alkaloid, a maytansinoid or maytansinoid analog such as DM1 or DM4, a cryptophycin derivative, an auristatin or dolastatin analog; a DNA alkylating agent, such as a tomaymycin or pyrrolobenzodiazepine derivative, a CC-1065 or CC-1065 analog ; a leptomycin derivative; a topoisomerase II inhibitor, an RNA polymerase II inhibitor such as alpha-amanitin.

According to a first embodiment, said at least one growth inhibitory agent is neither an undefined radioactive isotope, a chemotherapeutic drug, a protein or lectin, nor pokeweed antiviral protein, abrin, ricin and each of their A chains, doxorubicin, cisplastin, lodine-131 , Yttrium-90, Rhenium-188, Bismuth-212, Taxol, 5-Fluorouracil, VP-16 (etoposide), bleomycin, methotrexate, vindesine, adriamycin, vincristine, vinblastine, bis-chloroethylnitrosourea (BCNU), mitomycin, cyclophosphamide and a cytokine such as TNF and TNF-β.

According to this first embodiment, the invention relates in particular to an immunoconjugate comprising an antibody which:

a) binds to human and Macaca fascicularis LAMP1 proteins; and

b) is linked or conjugated to at least one growth inhibitory agents

(i) a cytotoxic agent selected from the group consisting of enzymes other than from pokeweed antiviral protein; antibiotics other than from bleomycin and mitomycin; toxins of bacterial, fungal, or animal origin or of plant origin other than from abrin and ricin, including fragments and/or

variants thereof ; a drug or a pro-drug of a compound consisting in an anti- tubulin agent such as a maytansinoid or maytansinoid analog such as DM1 or DM4, a taxoid or taxane other than from paclitaxel (Taxol), a vinca-alkaloid other than from vindesine, vincristine and vinblastine, a cryptophycin derivative, an auristatin or dolastatin analog ; a DNA alkylating agent other than from BCNU and cyclophosphamide, such as a tomaymycin or pyrrolobenzodiazepine derivative, a CC-1065 or CC-1065 analog ; a leptomycin derivative ; a topoisomerase II inhibitors other than doxorubicin (adriamycin) and etoposide, a RNA polymerase II inhibitor such as alpha-amanitin, or

(ii) a radioactive isotope selected from the group consisting of At211, Ac225, Bi213, Pb212, Er169, I124, I125, In111, P32, Re186, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu such as Lu177, and Th227. In one embodiment a radioactive isotope is selected from the group consisting of At211, Er169, I125, In111, P32, Re186, Sm153, Sr89, radioactive isotopes of Lu, and Th227.

In said first embodiment, the antibody may bind in particular to a domain consisting of the first to third loops of human and Macaca fascicularis LAMP1 proteins; wherein the domain consisting of the first to third loops of human LAMP1 protein consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and the domain consisting of the first to third loops of Macaca fascicularis LAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO: 39

According to a second embodiment, the invention relates to an immunoconjugate wherein the antibody binds to a domain consisting of the first to third loops of human and Macaca fascicularis LAMP1 proteins; wherein the domain consisting of the first to third loops of human LAMP1 protein consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and the domain consisting of the first to third loops of Macaca fascicularis LAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO: 39.

Therefore, according to this second embodiment, the immunoconjugate comprises an antibody which

a) binds to a domain consisting of the first to third loops of human and Macaca fascicularis LAMP1 proteins; wherein the domain consisting of the first to third loops of human LAMP1 protein consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and the domain consisting of the first to third loops of Macaca fascicularis LAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO: 39; and

b) is linked or conjugated to said at least one growth inhibitory agent.

Although not compulsory, in said second embodiment, the at least one growth inhibitory agent may be different from an undefined radioactive isotope, a chemotherapeutic drug, a protein or lectin, in particular from pokeweed antiviral protein, abrin, ricin and each of their A chains, doxorubicin, cisplastin, lodine-131 , Yttrium-90, Rhenium-188, Bismuth-212, Taxol, 5- Fluorouracil, VP-16 (etoiposide), bleomycin, methotrexate, vindesine, adriamycin, vincristine, vinblastine, BCNU, mitomycin, cyclophosphamide and a cytokine such as TNF and TNF-β.

Accordingly, said at least one growth inhibitory agent may be a radioactive isotopes selected from the group consisting of At211, Ac225, Bi213, Pb212, Er169, I124, I125, In111, P32, Re186, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu such as Lu177, and Th227, for instance At211, Er169, I125, In111, P32, Re186, Sm153, Sr89, radioactive isotopes of Lu such as Lu177, and Th227, or a cytotoxic agent as defined in said first embodiment.

In said first and second embodiments, said at least one growth inhibitory agent may be in particular drug or a pro-drug of a compound consisting in a maytansinoid or maytansinoid analog such as DM1 or DM4, a tomaymycin or pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or dolastatin analog, or a CC-1065 or CC-1065 analog, a RNA polymerase II inhibitor such as alpha-amanitin.

In one embodiment, a suitable tomamycin is a tomamycine dimer. Said tomamycin dimer is for instance (2E,2'E,1 1 aS,1 1 a'S)-8,8'-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)

bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1 Hbenzo[e]pyrrolo[1 ,2-a][1 ,4] diazepin-5(1 1 aH)-one).

The structural formula of (2E,2'E,1 1 aS,1 1 a'S)-8,8'-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)

bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1 Hbenzo[e]pyrrolo[1 ,2-a][1 ,4] diazepin-5(1 1 aH)-one) is

A "mavtansinoid" as used herein denotes maytansinoids and maytansinoid analogs. Maytansinoids are drugs that inhibit microtubule formation and that are highly toxic to mammalian cells.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs.

Examples of suitable maytansinol analogues include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331 ,598; 4,361 ,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371 ,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modified aromatic ring include:

(1 ) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of ansamytocin P2);

(2) C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (U.S. Pat. Nos. 4,361 ,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No 4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol having modifications of other positions include:

(1 ) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331 ,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.

4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudi flora);

(6) C-18-/V-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No 4,371 ,533) (prepared by the titanium trichloride/LAH reduction of maytansinol).

In a specific embodiment, the cytotoxic conjugates of the present invention utilize the thiol-containing maytansinoid (DM1 ), formally termed /V2-deacetyl-/V2-(3-mercapto-1 -oxopropyl)-maytansine, as the cytotoxic agent. DM1 is represented by the following structural formula (I):

ln another embodiment, the cytotoxic conjugates of the present invention utilize the thiol-containing maytansinoid DM4, formally termed /V2-deacetyl-/V2-(4-methyl-4-mercapto-1 -oxopentyl)-maytansine, as the cytotoxic agent. DM4 is represented by the following structural formula (II):

In further embodiments of the invention, other maytansines, including thiol and

disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom, may be used. These include a maytansinoid having, at C-3, C-14 hydroxy methyl, C-15 hydroxy, or C-20 desmethyl, an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has one or two substituents, said substituents being CH3, C2H5, linear or branched alkyl or alkenyl having from 1 to 10 reagents and any aggregate which may be present in the solution.

Examples of these cytotoxic agents and of methods of conjugation are further given in the application WO2008/010101 which is incorporated by reference.

In some embodiments of the present invention, the antibody is covalently attached, directly or via a cleavable or non-cleavable linker, to the at least one growth inhibitory agent.

"Linker", as used herein, means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a polypeptide to a drug moiety.

The conjugates may be prepared by in vitro methods. In order to link a drug or prodrug to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Conjugation of an antibody of the invention with cytotoxic agents or growth inhibitory agents may be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1 -pyrrolidinyl ester (nitro-SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), SNPP (N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate), succinimidyl (N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987). Carbon labeled 1 -isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO 94/1 1026).

The linker may be a "cleavable linker" facilitating release of the cytotoxic agent or growth inhibitory agent in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase labile linker, a photolabile linker or a disulfide-containing linker (See e.g. U.S. Patent No. 5,208,020) may be used. The linker may be also a "non-cleavable linker" (for example SMCC linker) that might lead to better tolerance in some cases.

Alternatively, a fusion protein comprising the antibody of the invention and a cytotoxic or growth inhibitory polypeptide may be made, by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
Genomic definition of LAMP1 (13q34) gain on Esophageal human tumor cance

In the Esophageal cancer DNA samples (Asterand), the LAMP1 gain or amplification is also including in a large amplicon, the largest gain region involves 4523kb (1 10584050 -1 15107245) and the smallest region present a LAMP1 amplification (Chr13q34) equal to 39.81 copy number. This focal amplification of LAMP1 covers 378 kb and includes 10 genes: ADPRHL1, CUL4A, F10, F7, GRTP1, LAMP1, LOC100130463, PCID2, PROZ and MCF2L.

Table 46: Description analysis of LAMP1 Copy number analysis of studied groups (LAMP1 Amplification, Gain, Diploid and Heteroloss (Complete or partial loss of one allele of LAMP1 gene) on Eosophagus cancer tissues.

Descriptive statistics for parameter Segment

(size in kb)

Standard

Status N Mean

Deviation

Loss 5 70415 29317.91

NoChange 39 48109.4 37656.46

Gain 1 4523

Amplification 1 378

Example 15: Relation between LAMP1 gene copy number and mRNA gene expression

Analyses of the mRNA expression level by gene expression profile and the copy number change at LAMP1 region

In addition to the analysis of LAMP1 Copy Number Change (amplification and gain), using the CRC tumors PDX, the correlation between LAMP1 amplification was evaluated by CGH analysis and LAMP1 expression by using mRNA (Affymetrix technology). Results from the mRNA analysis, using Pearson correlation test (Table 47) indicated high correlation ((r) = 0.59; p < 0.0001) between LAMP1 Copy Numbers and LAMP1 mRNA expression levels (Figure 8A). For the Colon tumors PDX, a Student test is performed to compare LAMP1 gene copy number (with or without gain/amplification) and LAMP1 mRNA expression. mRNA expression analysis was performed using Affymetrix technology (Table 48 and Figure 8B).

Table 47: LAMP1 : Copy Number Alteration Data and correlation with mRNA data on CRC PDX

Table 48: Student t-test of mRNA expression for factor copy number

The mRNA expression is significantly higher for LAMP1 Copy Numbers change (CN≥2.5).

The correlation analysis, using Pearson test between LAMP1 amplification by CGH analysis and LAMP1 expression by using mRNA, shows a significantly correlation between these two parameter studied.

As shown in Figure 8a, the group with LAMP1 high amplification (Amp) shows higher mRNA expression levels than groups with LAMP1 low amplification (Gain), Diploid and Hetloss.

The correlation analysis using a larger set of colorectal patients tissues samples

(n=574) from the TCGA (The Cancer Genome Atlas) data, disclosed 14.4% amplification that correlates with mRNA expression ((r) = 0.57; p<0.0001), this result is extremely similar with that observed on the CRC PDX.

Moreover, using the same dataset, a significant correlation of LAMP1 copy number change and mRNA expression level was evidenced for: Bladder Urothelial Carcinoma (BLCA), Breast Invasive Carcinoma (BRCA), Lung adenocarcinoma (LUAD), Lung squamous cell (LUSC) and Ovary (OV) (Figure 9).

Example 16: LAMP1 copy number variation and its impact on the LAMP1 protein cell membrane expression level

Association of LAMP1 copy number change and the protein cell membrane expression level detected by immunohistochemistry (IHC).

In addition to the analysis of LAMP1 gain and its relation with the LAMP1 RNA expression, we also evaluated association of LAMP1 copy number change (gain or amplification) to cell membrane LAMP1 protein localization, using IHC expression scoring (strong, medium, faint and negative) with antibody mAbl for colon, lung and stomach tumor PDXs.

As shown in tables 49 and 50 below, and figure 1 1 , analysis of IHC cell membrane expression in colon, lung and stomach PDXs samples shows that LAMP1 protein is expressed in the membrane cells in 39 out of 95 PDXs (41.1 %) models studied; 33 of these PDXs samples positive for LAMP1 membrane expression (33 out of 39; 85%) present also LAMP1 gain or amplification, most of these are Colon PDX.

Table 49: Frequency of LAMP1 Copy Number data and IHC scoring data of colon tumor PDX

Copy Number IHC

Frequency Neg_Faint Medium Strong Total

<2.5 15 4 0 19

>2.5 13 18 10 41

Total 28 22 10 60

Table 50: Frequency of LAMP1 Copy number data and IHC scoring data of lung and stomach tumor PDXs

Copy Number Tumor Type IHC

Frequency Neg_Faint Medium_Strong Total

Lung 9 2 1 1

Stomach 1 1 0 1 1

> ς Lung 5 1 6

Stomach 3 4 7

The association between IHC membrane expression and the copy number change was studied using Cochran-Mantel-Haenszel statistics (Tables 51 and 52).

Table 51 : Cochran-Mantel-Haenszel statistics of LAMP1 IHC membrane expression by the Copy Number of LAMP1 in the Colon PDX tumor samples.

In Colon tumor PDX, the association between LAMP1 IHC membrane expression and Copy Number of LAMP1 is significant.

Table 52: Cochran-Mantel-Haenszel statistics of LAMP1 IHC membrane expression by the Copy Number of LAMP1 in Lung and Stomach PDX tumor samples.

After adjusting for tumor type, the association between LAMP1 IHC membrane expression and Copy Number of LAMP1 is significant.

We conclude that the level of LAMP1 cell surface localization (Strong and medium) is associated with copy number change on tumor PDX samples. Most of cell surface localization of LAMP1 appears to be a consequence of LAMP1 gain or amplification.

Table 53: Table summarizing LAMP1 gene gain and LAMP1 expression

Example 17:- Specific peptide and mAb to detect LAMP1 membrane reinforcement on FFPE tumor tissue by immunohistochemistry (IHC)

IHC analysis of tumor tissues from biobanks or from hospitals before or during patient treatment is routinely done with formalin-fixed paraffin-embedded (FFPE) samples. Although commercially available mAbs and the three mAbs described previously (MAb1 , MAb2 and MAb3) can allow intracellular detection of LAMP1 and some of them, including MAb1 , 2 and 3, LAMP1 membrane reinforcement in frozen-OCT and AFA (Alcohol Formalin Acetic acid Fixative) sample format, none can lead to the detection of LAMP1 reinforcement in FFPE format. One of the reasons is probably the effect of the formalin fixative combined to the complexity of the protein. Samples processed in frozen OCT or AFA are not routinely prepared in hospitals. Therefore, there is a need to have a mAb that would allow complete and fast coverage of the FFPE tumor biobanks and hospital samples.

It is shown in the examples below that it was possible to overcome the difficulties by identifying a peptide (peptide 4) located in the second luminal domain at positions 360 to 375 of SEQ ID NO: 24, and having the amino acid sequence of SEQ ID NO: 82. Said peptide permited the obtention of rabbit polyclonal antibodies and mouse monoclonal antibody that led to the detection of LAMP1 membrane reinforcement in FFPE tissues.

Table 54 : List of antiLAMPI mAb tested and showing no LAMP1 membrane reinforcement on FFPE tissues by IHC

MAbs obtained from the following

Species clone number supplier

Epitomics Rabbit ERP4204

Novus Biologicals Mouse B-T47

Biolegend Mouse H4A3

United States Biol Mouse 5K76

Santa Cruz Mouse E-5

Santa Cruz Mouse H5G1 1

Biorbyt Mouse monoclonal

Biorbyt Rabbit monoclonal

MAbs described in this application

MAb1 Mouse monoclonal

MAb2 Mouse monoclonal

MAb3 Mouse monoclonal

Example 17.1 : Production of rabbit polyclonal antibodies that led to LAMP1 membrane reinforcement on FFPE tumor tissues

This example describes the selection of peptides in the human LAMP1 luminal domains, the generation of polyclonal antibodies and the IHC screening. It demonstrates the feasibility to obtain polyclonal antibodies that allow the detection of LAMP1 membrane reinforcement on formalin-fixed paraffin-embedded tissues when using the specific peptide ("peptide 4") of SEQ ID NO :82 corresponding to the amino acids at positions 360 to 375 on the human LAMP1 sequence of SEQ ID NO :24.

Example 17.1.1: Rabbit immunisation with peptides or soluble LAMP1 protein. Purification of polyclonal antibodies.

Peptide Preparation :

Peptides of 15-16 amino acids were selected within the two luminal domains without a N-glycosylation site and no internal cysteine. A total of four peptides were chemically synthesised and coupled to the Keyhole Limpet Hemocyanin (KLH) carrier protein. When needed, a terminal cysteine was previously added to the peptide so that coupling occurred via its thiol group to maleimide activated KLH protein.

Table 55 : Description of the four selected peptides

Immunisation and obtention of polyclonal antibodies.

A total of three programs of rabbit immunisations were performed. Rabbits were immunized in the first program, with peptide 1 SEQ ID NO : 90 and peptide 2 of SEQ ID NO : 91 , in the second program with peptide 4 of SEQ ID NO : 82 and peptide 3 of SEQ IOD NO : 92 and in the third program with heated denatured human LAMP1 ::histag protein produced as described in example 6.2. In brief, the immunisation schedule

comprised four injections and a final bleed aner 28 days. Polyclonal response was determined by ELISA on a sample from the final bleeds.

Purifications of polyclonal antibodies.

Reactive AF-aminoTOYOPEARL was used to couple each peptide described on Table 55 and to generate four affinity chromatography columns. The serum final bleeds on rabbits immunized with the respective peptides were purified by peptide affinity chromatography. The purified polyclonal batches were then characterized by SDS-PAGE and ELISA.

The serum final bleed on the rabbit immunized with LAMP1 protein was purified by protein G affinity chromatography. The purified polyclonal batch was then characterized by SDS-PAGE.

Example 17.1.2: IHC screening and identification of polyclonal rAb4 (rabbit Antibody 4) obtained by peptide 4 immunization

Rabbit polyclonal antibodies generated with peptides described in example 17.1.1 were tested by IHC on FFPE sample of colon adenocarcinoma patient derived xenograft CR-LRB-010P and human breast carcinoma. After antigen retrieval procedure and endogen biotins blocking steps, slides were incubated with the primary anti-antibody for 1 hour at 24°C. Negative controls were performed by omission of the primary antibody. The biotin free anti-rabbit UltraMap™ horseradish peroxidase (HRP) conjugate (760-4315, Ventana Medical Systems, Inc, USA) was used as secondary antibody system according to manufacturer's recommendations. Negative controls were performed by omission of the primary antibody. A couterstaining step was done with hematoxylin (760-2037, Ventana Medical Systems, Inc, USA) and bluing reagent was applied (760-2037, Ventana Medical Systems, Inc, USA). Stained slides were dehydrated and coverslipped with cytoseal XYL (8312-4, Richard-Allan Scientific, USA). Only antibodies from peptide 4 of SEQ ID NO : 82 immunization displayed LAMP1 membrane reinforcement in FFPE samples as shown in Figure 38 .

Example 17.1.3: Validation of polyclonal rabbit (rAb4) batch

ICC with cells expressing or not LAMP1 at the membrane

Human-LAMP1 and empty-vector HEK transfected cells were tested with the polyclonal rabbit rAb4 Antibody by immunocytochemistry (ICC) in FFPE format. High level of intracellular and surface cell LAMP1 immunostaining was obtained using the polyclonal rabbit rAb4 Antibody Ab at 1 μg mL as shown in Figure 39.

Affinity to LAMP1 protein

Secreted LAMP1 ::histag (29-382) with SEQ ID NO : 28 described in example 6.2 was used to determine the affinity of the polyclonal antibodies poly rAb4 by ELISA as described in example 6.3. The polyclonal rabbit antibody poly rAb4 binds to LAMP1 with an EC50 of around 3 nM whereas MAb1 binds with an EC50 of 0.16 nM.

Example 17.2: Obtention and characterization of mouse monoclonal antibodies that led to LAMP1 membrane reinforcement on FFPE tumor tissues

Example 17.1.1: Mouse immunisation and selection of mature IgG LAMP 1 -secreting hybridoma

While immunizations have been performed with diverse protein antigens including recombinant chimer human/ mouse LAMP1 protein, recombinant denatured human LAMP1 protein or recombinant human LAMP1 protein, these approaches were not successful in identifying antibody able to detect LAMP1 membrane reinforcement on FFPE tumor tissues. These approaches used immunization protocol described in example 2 for generation of anti-LAMP1 monoclonal antibodies and LAMP1 proteins described in example 6.2. On the contrary, the peptide 4-based immunization strategy has been shown to identify an antibody eligible to detect LAMP1 membrane reinforcement on FFPE tumor tissues.

Therefore mouse were immunised with peptide 4 and anti-LAMP1 -secreting hybridomas were selected as described below.

Generation of anti-LAMP1 monoclonal antibodies

Five BALB/cJ mice, about 6-8 weeks old (Charles River) were immunized with 40 μg of peptide peptide 4 of SEQ ID NO : 82 using RIMMS approach as described by Kilpatrick et al.; hybridoma, 1997: volume 16, number4. B cells immortalization using P3X63-AG8.653 (ATCC, ref CRL-1580) as fusion partner and hybridoma selection was performed as described in example 2.

Selection of anti-LAMPI antibodies by ELISA

The primary screen was an enzyme-linked immunosorbent assay (ELISA) assay (described in example 6.3 for anti-LAMP1 IgG production) using the LAMP1 ::histag protein described in example 6.2 as capturing antigen.

Example 17.2.2: IHC screening and identification of MAb4

As the same manner as in example 17.1.2, IHC screening was performed with the hybridoma supernatant to identify mouse monoclonal antibody showing LAMP1 membrane reinforcement on FFPE sample of colon adenocarcinoma patient derived xenograft CR-LRB-010P. The biotin free anti-mouse UltraMap™ horseradish peroxidase (HRP) conjugate (760-152, Ventana Medical Systems, Inc, USA) was used as secondary antibody system according to manufacturer's recommendations.

The supernatant of the selected hybridoma 88LAMP1 -2 displayed membrane reinforcement immunostaining in FFPE sample of colon adenocarcinoma patient derived xenograft CR-LRB-010P. Other irrelevant antibodies were negative or displayed intracellular immunostaining as shown in Figure 40.

Example 17.2.3: Validation of hybridoma 88LAMP1-2

Purification and characterisation of Mab4 obtained from hybridoma 88LAMP1-2

Hybridoma 88LAMP1 -2 was produced in medium A Clonacell-Hy (StemCell Technologies # 03801 ) supplemented with 5 % HCS (PAA; #F05-009) at the 400 ml_ scale and purified by protein A affinity chromatography. The purified antibody MAb4 was characterized by SDS-PAGE, and Mass Spectrometry. Masses of heavy and light chains from MAb4 were identified as reported in example 7 and are reported on the Table 55 below. Nucleic acid sequences encoding the variable domains were retrieved from hybridoma cells by RT-PCR as described in example 7. The corresponding amino acids from the heavy and light chains led to masses in agreement with the respective masses from MAb4.

Table 56: Mass characterization of MAb4

Example 17.3: In vitro characterisation of MAb4

Example 17.3.1 : Apparent affinity to human LAMP1 and cynomolgus LAMP1 by ELISA

Antibody MAb4 was assessed for its ability to bind primate LAMP1 protein by enzyme-linked immunosorbent assay (ELISA) assay as described in example 4.7 and EC50 values determined as described in example 6.2. Antibody MAb4 binds to human

LAMP1 and cynomolgus LAMP1 with similar affinity in range of 0.2 to 0.4 nM as shown in Table 57 below.

Table 57: EC50 determined by ELISA values on recombinant human LAMP1 and cynomolgus LAMP1

Example 17.3.2: Specificity to LAMP1

LAMP2 is the closest member of the LAMP family with 35 % sequence identity to LAMP1 . Specificity of MAb4 was evaluated by ELISA as described in example 4.6 with either LAMP1 or LAMP2 soluble proteins, as shown in Figure 41 . No binding to LAMP2 was detected with MAb4 and a difference of more than 100 fold is observed between the ECso of MAb4 towards LAMP1 versus LAMP2.

Example 17.3.3: Binding of antibody MAb4 to multiple cancer cells and determination of antibody binding capacity by Flow Cytometry

Antibody MAb4 was found to be able of binding to multiple tumor cells by Flow Cytometry using the conditions described in example 4.1. The panel of tumor cells comprises Patient-derived tumor xenografts from different origins and tumor cell lines. The Mean Flurescence Intensity (MFI) obtained from the flow cytometry analysis is reported in Table 58. Table 59 summarizes the antibody binding capacity results.

Table 58: Mean Florescence Intensity by FACS on Patient-derived xenografts

Mean Florescence

Intensity (MFI)

CR-IGR-034P / colorectal 424

LUN-NIC-006 / lung 162

LUN-NIC-033 / lung 154

BRE-IGR-0159 / breast 400

Colo205 / colon 7

Table 59: Antibody Binding Capacity by FACS on atient-derived xenograft

Example 17.3. 4: Apparent affinity of antibody MAb4 to human primary colon tumor PDX (CR-IGR-034P) by Flow Cytometry

Apparent affinity of antibody MAb4 was evaluated to human primary colon tumor PDX CR-IGR-034P by Flow Cytometry using the conditions described in example 4.1. ECso obtained with CR-IGR-034P with MAb4 was 1.3 nM.

Example 17.3. 5: IHC validation

Results obtained with purified batch are similar to those obtained in example 17.2.2 with none purified MAb4
CLAIMS

1 . An immunoconjugate comprising an antibody which:

a) binds to human and Macaca fascicularis LAMP1 proteins; and

b) is linked or conjugated to at least one growth inhibitory agent.

2. The immunoconjugate according to claim 1 , wherein said at least one growth inhibitory agent is

(i) a cytotoxic agent selected from the group consisting of enzymes other than pokeweed antiviral protein; antibiotics other than bleomycin and mitomycin; toxins of bacterial, fungal, or animal origin or of plant origin other than abrin and ricin, including fragments and/or variants thereof ; a drug or a pro-drug of a compound consisting in an anti-tubulin agent, a maytansinoid or maytansinoid analog, a taxoid or taxane other than paclitaxel (Taxol), a vinca-alkaloid other than vindesine, vincristine and vinblastine, a cryptophycin derivative, an auristatin or dolastatin analog; a DNA alkylating agent other than BCNU and cyclophosphamide, a tomaymycin or pyrrolobenzodiazepine derivative, a CC- 1065 or CC-1065 analog; a leptomycin derivative; a topoisomerase II inhibitors other than doxorubicin (adriamycin) and etoposide, a RNA polymerase II inhibitor, alpha-amanitin, or

(ii) a radioactive isotope selected from the group consisting of At211, Ac225, Bi2 3, Pb212, Er169, I124, I125, In111, P32, Re186, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu, and Th227.

3. The immunoconjugate according to claim 1 or 2, wherein at least 2 molecules of antibody according to the invention are internalized by one molecule of LAMP1 expressed at the surface of a cancer cell.

4. The immunoconjugate according to any one of claims 1 to 3, wherein said antibody binds to a domain consisting of first to third loops of human and Macaca fascicularis LAMP1 proteins; wherein the domain consisting of the first to third loops of human LAMP1 protein consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and the domain consisting of the first to third loops of Macaca fascicularis LAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO: 39.

5. The immunoconjugate according to any one of claims 1 to 4, wherein the antibody binds to a first lumenal domain of human and Macaca fascicularis LAMP1 proteins; wherein the first lumenal domain of human LAMP1 protein consists of amino acids at positions Ala29 to Arg195 of SEQ ID NO: 24 and the first lumenal domain of Macaca fascicularis LAMP1 protein consists of amino acids at positions Ala27 to Arg193 of SEQ ID NO: 39.

6. The immunoconjugate according to any one of claims 1 to 5, wherein the antibody competes for binding to the first lumenal domain of human and Macaca fascicularis LAMP1 proteins with an antibody comprising the variable heavy and light chains of

(i) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 1 and a variable domain of light chain of sequence of sequence SEQ ID NO: 5; or

(ii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 8 and a variable domain of light chain of sequence of sequence SEQ ID NO: 12; or

(iii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 15 and a variable domain of light chain of sequence of sequence SEQ ID NO: 16; or

(i) an antibody comprising a variable domain of heavy chain of sequence

SEQ ID NO: 42 and a variable domain of light chain of sequence of sequence SEQ ID NO: 46; or

(iv) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 42 and a variable domain of light chain of sequence of sequence SEQ ID NO: 51 ; or

(v) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 53 and a variable domain of light chain of sequence of sequence SEQ ID NO: 56; or

(vi) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 54 and a variable domain of light chain of sequence of sequence SEQ ID NO: 57; or

(vii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 55 and a variable domain of light chain of sequence of sequence SEQ ID NO: 58.

noconjugate according to any one of claims 1 to 6, wherein the antibody

a CDR1 -H of sequence SEQ ID NO: 2 or a sequence differing from SEQ ID NO: 2 by one amino acid substitution, a CDR2-H of sequence SEQ ID NO: 3 or a sequence differing from SEQ ID NO: 3 by one amino acid substitution, and a CDR3-H of sequence SEQ ID NO: 4 or a sequence differing from SEQ ID NO: 4 by one amino acid substitution; or a CDR1 -L of sequence SEQ ID NO: 6 or a sequence differing from SEQ ID NO: 6 by one amino acid substitution, a CDR2-L of sequence DTS or a sequence differing from DTS by one amino acid substitution and a CDR3-L of sequence SEQ ID NO: 7 or a sequence differing from SEQ ID NO: 7 by one amino acid substitution; or

a CDR1 -H of sequence SEQ ID NO: 9 or a sequence differing from SEQ ID NO: 9 by one amino acid substitution, a CDR2-H of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution, a CDR3-H of sequence SEQ ID NO: 1 1 or a sequence differing from SEQ ID NO: 1 1 by one amino acid substitution; or a CDR1 -L of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution, a CDR2-L of sequence AAS or a sequence differing from AAS by one amino acid substitution, and a CDR3-L of sequence SEQ ID NO: 14 or a sequence differing from SEQ ID NO: 14 by one amino acid substitution; or

a CDR1 -H of sequence SEQ ID NO: 43 or a sequence differing from SEQ ID NO: 43 by one amino acid substitution, a CDR2-H of sequence SEQ ID NO: 44 or a sequence differing from SEQ ID NO: 44 by one amino acid substitution, and a CDR3-H of sequence SEQ ID NO: 45 or a sequence differing from SEQ ID NO: 45 by one amino acid substitution; or

a CDR1 -L of sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution, a CDR2-L of sequence YTS or a sequence differing from YTS by one amino acid substitution, and a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52 or a sequence differing from SEQ ID NO: 48 or SEQ ID NO: 52 by one amino acid substitution.

8. The immunoconjugate according to claim 7, wherein said amino acid substitution is a conservative amino acid substitution.

9. The immunoconjugate according to any one of claims 1 to 8, wherein the antibody comprises:

(i) a CDR1 -H of sequence SEQ ID NO: 2, a CDR2-H of sequence SEQ ID NO: 3, a CDR3-H of sequence SEQ ID NO: 4, a CDR1 -L of sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L of sequence SEQ ID NO: 7; or

(ii) a CDR1 -H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ ID NO: 10, a CDR3-H of sequence SEQ ID NO: 1 1 , a CDR1 -L of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS, and a CDR3-L of sequence SEQ ID NO: 14; or

(iii) a CDR1 -H of sequence SEQ ID NO: 43, a CDR2-H of sequence SEQ ID NO: 44, a CDR3-H of sequence SEQ ID NO: 45, a CDR1 -L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52; or

(iv) a fragment of an antibody as defined in (i), (ii), or (iii).

10. The immunoconjugate according to any one of claims 1 to 9, wherein the antibody comprises:

(i) a variable domain of heavy chain of sequence SEQ ID NO: 1 or a sequence at least 85% identical thereto or a variable domain of light chain of sequence of sequence SEQ ID NO: 5, or a sequence at least 85% identical thereto; or

(ii) a variable domain of heavy chain of sequence SEQ ID NO: 8, or a sequence at least 85% identical thereto, or a variable domain of light chain of sequence of sequence SEQ ID NO: 12, or a sequence at least 85% identical thereto; or

(iii) a variable domain of heavy chain of sequence SEQ ID NO: 15, or a sequence at least 85% identical thereto, or a variable domain of light chain of sequence of sequence SEQ ID NO: 16, or a sequence at least 85% identical thereto; or

(iv) a variable domain of heavy chain of sequence SEQ ID NO: 42, or a sequence at least 85% identical thereto, or a variable domain of light chain of sequence of sequence SEQ ID NO: 46 or SEQ ID NO: 51 , or a sequence at least 85% identical thereto, or

(v) a variable domain of heavy chain of sequence SEQ ID NO: 53 or a sequence at least 85% identical thereto and/or a variable domain of light chain of sequence of sequence SEQ ID NO: 56, or a sequence at least 85% identical thereto; or

(vi) a variable domain of heavy chain of sequence SEQ ID NO: 54 or a sequence at least 85% identical thereto or a variable domain of light chain of sequence of sequence SEQ ID NO: 57, or a sequence at least 85% identical thereto; or

(vii) a variable domain of heavy chain of sequence SEQ ID NO: 55 or a sequence at least 85% identical thereto or a variable domain of light chain of sequence of sequence SEQ ID NO: 58, or a sequence at least 85% identical thereto.

1 1 . The immunoconjugate according to any one of claims 1 to 10, wherein the antibody does not significantly cross-react with human LAMP2 (SEQ ID NO: 40).

12. The immunoconjugate according to any one of claims 1 to 1 1 , wherein the antibody has an affinity measured via flow cytometry for human LAMP 1 and Macaca fascicularis LAMP1 expressed on the cell surface of a recombinant cell line, which is < 35nM.

13. The immunoconjugate according to any one of claims 1 to 12 wherein the antibody is a chimeric or a humanised antibody.

14. The immunoconjugate according to any one of claims 1 to 13, wherein the antibody comprises:

i) a heavy chain of sequence SEQ ID NO: 17 or a light chain of sequence SEQ ID NO: 18; or

ii) a heavy chain of sequence SEQ ID NO: 19 or a light chain of sequence SEQ

ID NO: 20; or

iii) a heavy chain of sequence SEQ ID NO: 21 or a light chain of sequence SEQ ID NO: 22; or

iv) a heavy chain of sequence SEQ ID NO: 49 or a light chain of sequence SEQ ID NO: 50,or

v) a heavy chain of sequence SEQ ID NO: 49 or a light chain of sequence SEQ ID NO: 81 ,or

vi) a heavy chain of sequence SEQ ID NO: 60 or a light chain of sequence SEQ ID NO: 59; or

vii) a heavy chain of sequence SEQ ID NO: 62 or a light chain of sequence SEQ

ID NO: 61 ; or

Viii) a heavy chain of sequence SEQ ID NO: 64 or a light chain of sequence SEQ ID NO: 63.

15. The immunoconjugate according to any one of claims 1 to 3, wherein the antibody binds to the fourth loop of human and Macaca fascicularis LAMP1 proteins; wherein the fourth loop of human LAMP1 protein consists of amino acids at positions Leu310 to Met382 of SEQ ID NO: 24 and the fourth loop of Macaca fascicularis LAMP1 protein consists of amino acids at positions Leu 308 to Met380 of SEQ ID NO: 39.

16. The immunoconjugate according to claim 15 wherein the antibody binds to a region of Loop 4 comprising the amino acids 360 to 375 of human LAMP1 that consists of sequences SEQ ID NO: 82.

17. The immunoconjugate according to claims 15 or 16, wherein the antibody comprises a CDR1 -H of sequence SEQ ID NO: 83, a CDR2-H of sequence SEQ ID NO: 84, a CDR3-H of sequence SEQ ID NO: 85, a CDR1 -L of sequence SEQ ID NO: 86, a CDR2-L of sequence NAK, and a CDR3-L of sequence SEQ ID NO: 87.

18. The immunoconjugate according to any one of claims 15 to 17, wherein the antibody comprises a variable domain of heavy chain of sequence SEQ ID NO: 88 or a sequence at least 85% identical thereto or a variable domain of light chain of sequence of sequence SEQ ID NO: 89, or a sequence at least 85% identical thereto.

19. The immunoconjugate according to any one of claims 15 to 18, wherein the antibody is a chimeric or a humanised antibody.

20. The immunoconjugate according to any one of claims 1 to 19, wherein the antibody is an antibody fragment.

21 . The immunoconjugate according to any one of claims 1 to 20, wherein the antibody is a fragment selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies and VHH.

22. The immunoconjugate according to any one of claims 1 to 21 , wherein the antibody is a bispecific or a multispecific antibody.

23. The immunoconjugate according to any one of claims 1 to 22, wherein said at least one growth inhibitory agent is a cytotoxic agent or a radioactive isotope.

24. The immunoconjugate according to any one of claims 1 and 2 to 23, wherein said at least one growth inhibitory agent is selected from the group consisting of chemotherapeutic agents, enzymes, antibiotics, toxins, taxoids, vincas, taxanes, maytansinoid or maytansinoid analogs, tomaymycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, and CC-1065 or CC-1065 analogs.

25. The immunoconjugate according to any one of claims 1 and 2 to 23, wherein said at least one growth inhibitory agent is selected from the group consisting of At211, Ac225,

Bi212, Er169, I131, I124, I125, Y90, In111, P32, Re186, Re188, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64, radioactive isotopes of Lu, and Th227.

26. The immunoconjugate according to any one of claims 1 to 25, wherein said at least one growth inhibitory agent is

(i) a cytotoxic agent selected from the group consisting of enzymes other than from pokeweed antiviral protein; antibiotics other than from bleomycin and mitomycin; toxins of bacterial, fungal, or animal origin or of plant origin other than from abrin and ricin, including fragments and/or variants thereof; a drug or a pro- drug of a compound consisting in an anti-tubulin agent,a maytansinoid or maytansinoid analog, a taxoid or taxane other than from paclitaxel (Taxol), a vinca-alkaloid other than from vindesine, vincristine and vinblastine, a cryptophycin derivative, an auristatin or dolastatin analog; a DNA alkylating agent other than from BCNU and cyclophosphamide, a tomaymycin or pyrrolobenzodiazepine derivative, a CC-1065 or CC-1065 analog; a leptomycin

derivative ; a, topoisomerase II inhibitors other than doxorubicin (adriamycin) and etoposide, a RNA polymerase II inhibitor, alpha-amanitin,or

(ii) a radioactive isotopes selected from the group consisting of At211, Ac225, Bi213, Pb212, Er169, I124, I125, In111, P32, Re186, Sm153, Sr89, Zr89, Tc99m, Ga68, Cu64 and radioactive isotopes of Lu such as Lu177, and Th227.

27. The immunoconjugate conjugate according to any one of claims 1 to 26, wherein said growth inhibitory agent is (/V2-deacetyl-/V2-(3-mercapto-1 -oxopropyl)-maytansine) DM1 or /V2-deacetyl-/V-2 (4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4).

28. The immunoconjugate according to any one of the claims 1 to 27, wherein the antibody is covalently attached via a cleavable or non-cleavable linker to the at least one growth inhibitory agent.

29. The immunoconjugate according to claim 28, wherein said linker is selected from the group consisting of N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl (N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).

30. The immunoconjugate according to claim 29, wherein said linker is N-succinimidyl pyridyldithiobutyrate (SPDB) and the growth inhibitory agent is /V2-deacetyl-/V2-(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4).

31 . The immunoconjugate according to claim 30, wherein said linker is 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB) and the growth inhibitory agent is N2-deacetyl-/V2-(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4).

32. The immunoconjugate according to claim 29, wherein said linker is succinimidyl (N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC) and the growth inhibitory agent is /V2-deacetyl-/V2-(3-mercapto-1 -oxopropyl)-maytansine (DM1 ).

33. The immunoconjugate according to any one of claims 27 to 32, wherein the immunoconjugate is characterised by a drug-to-antibody ratio (DAR) ranging from 1 to 10, preferably between 2 to 5, more preferably between 3 to 4, the DAR being calculated from the ratio of the drug concentration to that of the antibody :

DAR = cD / cA

wherein

CD = [(SA280 X A252) - (SA252 X A280)] / [(SD252 X SA28O) " (SA252 X SD28O)]

CA = [A28O - (CD X SD28o)] SA280

and

SD252 and SD28O are respectively the molar extinction coefficients of the drug at 252 nm and 280 nm;

SA252 and SA28O are respectively the molar extinction coefficients of the antibody at 252 nm and 280 nm;

(A252) and A28o are respectively the absorbances for the conjugate at 252 nm (A252) and at 280 nm (Α2βο), measured using a classic spectrophotometer apparatus.

34. An isolated antibody which binds to the first lumenal domain of human and Macaca fascicularis LAMP1 proteins, wherein the first lumenal domain of human LAMP1 is defined by the amino acids at positions Ala29 to Arg195 of SEQ ID NO: 24, and the first lumenal domain of Macaca fascicularis LAMP1 protein is defined by the amino acids at positions Ala27 to Arg193 of SEQ ID NO: 39.

35. The isolated antibody according to claim 34, which competes for binding to the first lumenal domain of human and Macaca fascicularis LAMP1 proteins with an antibody comprising the variable heavy and light chains of

(i) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 1 and a variable domain of light chain of sequence of sequence SEQ ID NO: 5; or

(ii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 8 and a variable domain of light chain of sequence of sequence SEQ ID NO: 12; or

(iii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 15 and a variable domain of light chain of sequence of sequence SEQ ID NO: 16; or

(ii) an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 42 and a variable domain of light chain of sequence of sequence SEQ ID NO: 46; or

an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 42 and a variable domain of light chain of sequence of sequence SEQ ID NO: 51 ; or

an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 53 and a variable domain of light chain of sequence of sequence SEQ ID NO: 56, or

an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 54 and a variable domain of light chain of sequence of sequence SEQ ID NO: 57, or

an antibody comprising a variable domain of heavy chain of sequence SEQ ID NO: 55 and a variable domain of light chain of sequence of sequence SEQ ID NO: 58.

36. The isolated antibody according to claim 34 or 35, which comprises

(i) a CDR1 -H of sequence SEQ ID NO: 2 , a CDR2-H of sequence SEQ ID

NO: 3, and a CDR3-H of sequence SEQ ID NO: 4; or

a CDR1 -L of sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L of sequence SEQ ID NO: 7; or

(ii) a CDR1 -H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ ID NO: 10, a CDR3-H of sequence SEQ ID NO: 1 1 ; or

a CDR1 -L of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS, and a CDR3-L of sequence SEQ ID NO: 14; or

(iii) a CDR1 -H of sequence SEQ ID NO: 43, a CDR2-H of sequence SEQ ID NO: 44, and a CDR3-H of sequence SEQ ID NO: 45, or a CDR1 -L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52.

37. The isolated antibody according to any one of claims 34 to 36, which is as defined in claims 5 to 15.

38. An isolated antibody which binds to the fourth loop of human and Macaca fascicularis LAMP1 proteins; wherein the fourth loop of human LAMP1 protein consists of amino acids at positions Leu310 to Met382 of SEQ ID NO: 24 and the fourth loop of Macaca fascicularis LAMP1 protein consists of amino acids at positions Leu 308 to Met380 of SEQ ID NO: 39.

39. The isolated antibody according to claim 38, wherein the antibody binds to a region of Loop 4 comprising the amino acids 360 to 375 of human LAMP1 that consists of sequences SEQ ID NO: 82.

40. The isolated antibody according to claim 38 or 39, wherein the antibody comprises a CDR1 -H of sequence SEQ ID NO: 83, a CDR2-H of sequence SEQ ID NO: 84, a CDR3-H of sequence SEQ ID NO: 85, or a CDR1 -L of sequence SEQ ID NO: 86, a CDR2-L of sequence NAK, and a CDR3-L of sequence SEQ ID NO: 87.

41 . The isolated antibody according to any one of claims 38 to 40, wherein the antibody comprises a variable domain of heavy chain of sequence SEQ ID NO: 88 or a sequence at least 85% identical thereto ora variable domain of light chain of sequence of sequence SEQ ID NO: 89, or a sequence at least 85% identical thereto.

42. An isolated anti-LAMP-1 antibody which comprises:

(vii) a CDR1 -H of sequence SEQ ID NO: 2 , a CDR2-H of sequence SEQ ID NO: 3, and a CDR3-H of sequence SEQ ID NO: 4; or

a CDR1 -L of sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L of sequence SEQ ID NO: 7; or

(viii) a CDR1 -H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ ID NO: 10, a CDR3-H of sequence SEQ ID NO: 1 1 ; or

a CDR1 -L of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS, and a CDR3-L of sequence SEQ ID NO: 14; or

(ix) a CDR1 -H of sequence SEQ ID NO: 43, a CDR2-H of sequence SEQ ID

NO: 44, and a CDR3-H of sequence SEQ ID NO: 45, or a CDR1 -L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52; or

(x) CDR1 -H of sequence SEQ ID NO: 83, a CDR2-H of sequence SEQ ID NO: 84, a CDR3-H of sequence SEQ ID NO: 85, or a CDR1 -L of sequence SEQ ID NO: 86, a CDR2-L of sequence NAK, and a CDR3-L of sequence SEQ ID NO: 87; ora heavy chain of sequence SEQ ID NO: 60 or a light chain of sequence SEQ ID NO: 59; or

(xi) a heavy chain of sequence SEQ ID NO: 62 or a light chain of sequence SEQ ID NO: 61 ; or

(xii) a heavy chain of sequence SEQ ID NO: 64 or a light chain of sequence SEQ ID NO: 63.

43. The isolated antibody according to any one of claims 38 to 42, wherein said antibody is labelled with a detectable molecule or substance.

44. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 1 to 33, or an antibody according to any one of claims 34 to 42, and a pharmaceutically acceptable carrier.

45. An immunoconjugate according to any one of claims 1 to 34, or an antibody according to any one of claims 34 to 42, or a pharmaceutical composition according to claim 44, for use for the treatment of cancer.

46. The immunoconjugate, antibody or pharmaceutical composition for the use according to claim 45, wherein the cancer is colon cancer adenocarcinoma, gastrointestinal tumors, vital organs tumors, reproductive organ tumors, or skin, larynx or soft tissue tumor.

47. An antibody according to any one of claims 34 to 43 for use for ex vivo detecting LAMP1 expression in biological sample of a subject.

48. An antibody according to any one of claims 34 to 43 for use for in vivo detecting LAMP1 expression in a subject.

49. The antibody for the use according to claim 47 or 48, wherein said antibody is labelled with a detectable molecule or substance.

50. The antibody for the use according to any one of claims 46 to 49, wherein said use is for diagnosing the presence of a cancer in a subject, determining susceptibility of a patient having cancer to a therapeutic agent targeting LAMP1 , or monitoring effectiveness of anti-LAMP1 cancer therapy or detecting cancer relapse after anti-LAMP1 cancer therapy.

51 . An isolated nucleic acid comprising a sequence encoding an antibody according to any one of claims 34 to 42.

52. A host cell which has been transformed by a nucleic acid according to claim 51.

53. An in vitro method of selecting patients with cancer who are likely to respond to anti-LAMP1 therapy, wherein said method comprises:

a. determining, in a biological sample of a patient with cancer which includes cancer cells, if said patient harbors a LAMP1 gene copy number gain; and

b. selecting the patient based on the presence of LAMP1 gene copy number gain,

and wherein said patient is selected as likely to respond to anti-LAMP1 therapy if said patient harbors a LAMP1 gene copy number gain.

54. The method according to claim 53, which further comprises determining if LAMP1 is expressed at the surface of cancer cells of the patient, and wherein i) said patient is selected as likely to respond to anti-LAMP1 therapy if said patient harbors a LAMP1 gene copy number gain and if said cancer cells of the patient express LAMP1 at their surface.

55. The method according to claim 53 or 54, wherein said cancer is selected from the group consisting of bladder, breast, cervical, colorectal, glioblastoma, head and neck, kidney, liver, lung, glioma, ovarian, pancreatic, prostate, stomach, thyroid, and uterine cancer.

56. The method according to any one of claims 53 to 55, wherein said cancer is selected from the group consisting of bladder, cervical, colorectal, glioblastoma, head and neck, kidney, liver, lung, glioma, ovarian, pancreatic, prostate, stomach, thyroid, and uterine cancer.

57. The method according to any one of claims 53 to 56, wherein said cancer is colorectal or lung cancer.

58. A method according to any one of claims 53 to 57, wherein mean LAMP1 gene copy number in cancer cells is≥ 2.5.

59. Method according to any one of claims 53 to 58, wherein mean LAMP1 gene copy

number in cancer cells is > 5.

60. A method according to any one of claims 53 to 59, wherein LAMP1 gene copy number gain is determined with a method selected from the group consisting of Fluorescence In Situ Hybridization (FISH), Comparative Genomic Hybridization (CGH), New Generation Sequencing (NGS) or Polymerase Chain Reaction (PCR).

61 . A method according to claim 60, wherein LAMP1 gene copy number gain is determined by Comparative Genomic Hybridization (CGH).

62. A method according to claim 60, wherein LAMP1 gene copy number gain is determined by Polymerase Chain Reaction (PCR).

63. A method according to claim 60, wherein LAMP1 gene copy number gain is determined by Fluorescence In Situ Hybridization (FISH).

64. A method according to claim 60, wherein LAMP1 gene copy number gain is determined by New Generation Sequencing (NGS).

65. An anti-LAMP1 therapeutic agent for use for treating cancer in a patient harboring LAMP1 gene copy number gain in cancer cells.

66. The anti-LAMP1 therapeutic agent for the use according to claim 65, wherein said patient harboring LAMP1 gene copy number gain in cancer cells has been selected by a method according to any one of claims 53 to 64.

67. The anti-LAMP1 therapeutic agent for the use according to claim 65, which use comprises selecting said patient harboring LAMP1 gene copy number gain in cancer cells by a method according to any one of claims 53 to 64.

68. The anti-LAMP1 therapeutic agent for the use according to any one of claims 65 to 67, wherein said anti-LAMP1 therapeutic agent is an anti-LAMP1 antibody according to any one of claims 34 to 42 or an immunoconjugate comprising an anti-LAMP1 antibody and at least one growth inhibitory agent according to any one of claims 1 to 33.