DESC:RELATED APPLICATIONS
This application is related to Indian provisional Application 202221044102 filed on 2nd Aug, 2022 and is incorporated herein in its entirely.

FIELD OF THE INVENTION

The present invention relates to lentiviral vectors consist of sequence encoding chimeric antigen receptor which are useful for the treatment of CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia.

BACKGROUND OF THE INVENTION

The large majority of patients having B-cell malignancies, including chronic lymphocytic leukemia (CLL), will die from their disease. One approach to treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs). CARs are antigen receptors that are designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts in using genetically modified cells expressing CARs to treat these types of patients have met with very limited success.

The CD19 molecule is a receptor for specific signal transduction on the surface of B lymphocytes, which exists in various stages of B cell maturation, appears in the B progenitor cell stage and is stably and continuously expressed. As a B cell line-specific cell surface differentiation antigen, CD19 is expressed only on the surface of pre-B cells and mature B cells, but not in hematopoietic stem cells, plasma cells and other normal tissue cells. Moreover, CD19 molecules are relatively exposed on the membrane, are easily accessible, have no significant internalization and shedding after binding with monoclonal antibodies, and do not undergo antigen modulation due to binding to antibodies, so CD19 is one of the most reliable surface of B cells.

CD19 regulates signaling through B cell receptors, which play an important role in B cell development, proliferation and differentiation, and malignant transformation. Based on the above phenomena, CD19 is an important target for the treatment of cells that develop and develop CD19 positive B-cell malignancies. CD19 has high levels of expression in most B cell lymphomas (such as DLBCL, FL, and mantle cell lymphoma), acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and a subset of acute myeloid leukemia while it is absent in hematopoietic stem cells, plasma cells and other normal human tissues. Therefore, the targeted treatment of CD19 does not cross-react with other normal tissues.

In most cancers, tumor-specific antigens are not yet well defined, but in B cell malignancies, CD 19 is an attractive tumor target. Expression of CD19 is restricted to normal and malignant B cells, so that CD19 is a widely accepted target to safely test CARs. While CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion and disappointing clinical activity.

CD19-targeted CAR T cells have emerged as a highly effective therapy in patients with refractory B-cell malignancies. Genetically modifying autologous T cells to express an anti- CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials. A CAR is a recombinant receptor construct composed of an antibody-derived extracellular single-chain variable fragment (scFv), linked to intracellular T-cell signaling domains of the T-cell receptor (TCR), thereby redirecting T-cell specificity to the tumor. The extracellular region responsible for binding to a particular antigen and an intracellular region that promotes T cell cytotoxic activity and proliferation.

Thus, there is an urgent need in the art for compositions and methods for treatment of cancer using CARs. The present invention addresses this need.

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequences encoding chimeric antigen receptor against CD19.

Another object of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another object of the present invention is to generate CAR-T cells by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain and human 4-1BB and CD3 zeta signaling domains, into the cells. The CAR-T cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

Another object of the present invention is to provide an isolated to purified nucleic acid sequences encoding chimeric antigen receptor against CD19; wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta as T-cell signaling domain.

Another object of the present invention is to provide an isolated or purified nucleic acid sequences having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19; wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta as T-cell signaling domain.
Another object of the present invention is to provide method for recombinant lentivirus (LV) particles preparation whereas, the lentivirus particle is packed with genes expressing chimeric antigens receptors.
Another object of the present invention is to provide viral genome of recombinant lentivirus (LV) particle which comprises following elements:-
(a) One or more left and right long terminal repeat (LTR) sequences in its native or truncated form, that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;
(b) HIV1 psi sequence;
(c) RRE i.e. Rev response element of HIV1;
(d) HIV central polypurine tract (cPPT) / central termination sequence (CTS);
(e) EF1? promoter; and
(f) WPRE element.
Another object of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR1/CAR2/CAR3/CAR4/CAR5/CAR6/CAR7/CAR9;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.

Another object of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR6;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.
Another object of the present invention to provide CAR-T cell for the treatment of CD19+ B-cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia.

Another object of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another object of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having at least 85% identity to the nucleotide sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequences encoding chimeric antigen receptor against CD19.

Another aspect of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another aspect of the present invention is to generate CAR-T cells by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain and human 4-1BB and CD3 zeta signaling domains, into the cells. The CAR-T cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

Another aspect of the present invention is to provide an isolated to purified nucleic acid sequences encoding chimeric antigen receptor against CD19; wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta as T-cell signaling domain.

Another aspect of the present invention is to provide an isolated or purified nucleic acid sequences having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19; wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta as T-cell signaling domain.
Another aspect of the present invention is to provide method for recombinant lentivirus (LV) particles preparation whereas, the lentivirus particle is packed with genes expressing chimeric antigens receptors.
Another aspect of the present invention is to provide viral genome of recombinant lentivirus (LV) particle which comprises following elements:-
(a) One or more left and right long terminal repeat (LTR) sequences in its native or truncated form, that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;
(b) HIV1 psi sequence;
(c) RRE i.e. Rev response element of HIV1;
(d) HIV central polypurine tract (cPPT) / central termination sequence (CTS);
(e) EF1? promoter; and
(f) WPRE element.
Another aspect of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR1/CAR2/CAR3/CAR4/CAR5/CAR6/CAR7/CAR9;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.

Another aspect of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR6;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.
Another aspect of the present invention to provide CAR-T cell for the treatment of CD19+ B-cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia.

Another aspect of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another aspect of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having at least 85% identity to the nucleotide sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

BRIEF DESCRIPTION OF DRAWINGS
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

Figure 1: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR1) & (B) Expression cassette design of anti-CD19 CAR1 with Transgene and Regulatory Elements.
Figure 2: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR2) & (B) Expression cassette design of anti-CD19 CAR2 with Transgene and Regulatory Elements.
Figure 3: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR3) & (B) Expression cassette design of anti-CD19 CAR3 with Transgene and Regulatory Elements.
Figure 4: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR4) & (B) Expression cassette design of anti-CD19 CAR4 with Transgene and Regulatory Elements.
Figure 5: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR5) & (B) Expression cassette design of anti-CD19 CAR5 with Transgene and Regulatory Elements.
Figure 6: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR6) & (B) Expression cassette design of anti-CD19 CAR6 with Transgene and Regulatory Elements.
Figure 7: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR7) & (B) Expression cassette design of anti-CD19 CAR7 with Transgene and Regulatory Elements.
Figure 8: Schematic Representation of (A) Vector Map (pIntas-EF-1?-anti-CD19 CAR9) & (B) Expression cassette design of anti-CD19 CAR9 with Transgene and Regulatory Elements.
Figure 9: Representative depiction of CAR T cell phenotype; (A) dragged (CAR1, CAR3, CAR5 and CAR9) vs. (B) compact (CAR2, CAR4, CAR6 and CAR7) based on spread in flow cytometry.
Figure 10: CAR T positive cell seven days post transduction with respective CAR LV at 0.3 MOI (A) and CD19 non-specific (K562; B and C) and CD19-specific (Raji. D and E) target cell killing at effector vs. target ratios (E : T) 3 : 1 (B and D) and 6 : 1 (C and E). CD19-specific killing is defined as value obtained by subtracting % cytotoxicity of K562 cells from % cytotoxicity of Raji cells. Significance was determined compared to CAR1. *, ** and *** depicts p-value significance at 0.05, 0.01 and 0.001, respectively.
Figure 11: (A) Cytotoxicity exerted by CAR1, CAR3, CAR6 and CAR9 on CD19 specific (Raji) and nonspecific (K562) at different ratios of co-culture; T+CAR-T cell exhaustion of CD4 (B) and CD8 (C) cells on the day of harvest (Day 12).

Figure 12: (A) Cytotoxicity of the T/CAR T cells generated for POC study in 24 well plate at a ratio of 3:1 (E:T); (B) Cytokine release profile analyzed by multiplex from the co-culture supernatant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for treating cancer. The present invention relates to a strategy of adoptive cell transfer of T cells transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity.

The lentiviral vector are designed to express a chimeric antigen receptor against CD19 and the vector comprising such nucleic acid. These CAR are well suited for treatment of CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and a subset of acute myeloid leukemia.

The recombinant lentivirus particles are prepared whereas, the lentivirus (LV) particles is packed with genes expressing chimeric antigen receptors. Such viral vectors can be used for the treatment of B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and a subset of acute myeloid leukemia by using administration of nucleotide sequence.

DEFINITION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.

The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of” or “consisting essentially” of language.

The term "antibody" as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immune-reactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv fragments, scFv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multi-specific antibodies formed from antibody fragments.

An "antibody heavy chain" as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

An "antibody light chain" as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations and ? light chains refer to the two major antibody light chain isotype.

A "co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation, Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A "co-stimulatory signal" as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or up-regulation or down-regulation of key molecules.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA or an RNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

A "vector" is a composition of matter which comprises an isolated nucleic acid & which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in 5 the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids & viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, poly lysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, Adeno-associated virus vectors, retroviral vectors, and the like.

A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses.

The term "viral vector" may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and Adeno-associated viruses) that incorporate the recombinant polynucleotide.

A "lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, S1V and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

By the term "stimulation" is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex, Stimulation can mediate altered expression of certain molecules, such as down-regulation of TGF- ß, and/or reorganization of cytoskeletal structures, and the like.

To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The main embodiment of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequences encoding chimeric antigen receptor against CD19.

Another embodiment of the present invention is to provide lentivirus (LV) vector comprising nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another embodiment of the present invention is to generate CAR-T cells by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain, and human 4-1BB and CD3 zeta signaling domains, into the cells. The CAR T cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

Another embodiment of the present invention is to provide an isolated or purified nucleic acid sequences encoding chimeric antigen receptor against CD19, wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta T-cell signaling domain.

Another embodiment of the present invention is to provide an isolated or purified nucleic acid sequences having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19; wherein the chimeric antigen receptor sequence comprises following elements:-
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 41BB and/or CD28; and
(f) CD3 zeta T-cell signaling domain.

Another embodiment of the present invention is to provide methods for recombinant lentivirus (LV) particles preparation whereas, the lentivirus particle is packed with genes expressing chimeric antigens receptors.

Another embodiment of the present invention is to provide viral genome of recombinant lentivirus (LV) particle which comprises following elements:-
(a) One or more left and right long terminal repeat (LTR) sequences in its native or truncated form, that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;
(b) HIV1 psi sequence;
(c) RRE i.e. Rev response element of HIV1;
(d) HIV central polypurine tract (cPPT) / central termination sequence (CTS);
(e) EF1? promoter; and
(f) WPRE element.

Another embodiments of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR1/CAR2/CAR3/CAR4/CAR5/CAR6/CAR7/CAR9;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.

Another embodiments of the present invention is to provide recombinant lentivirus (LV) vector, wherein said viral vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR6;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.

In one embodiment of the present invention 5’ LTR, HIV-1 psi, RRE, cPPT/CTS, EF-1? promoter, WPRE and 3’ LTR (Delta-U3) are having nucleic acid sequence of SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 respectively.
Another embodiment of the present invention to provide CAR-T cell for treatment of CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia.
In another embodiment of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequences having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

Another embodiment of the present invention is to provide method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having at least 85% identity to the nucleotide sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.

The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXPERIMENTAL DETAILS
EXAMPLE 1: CAR CONSTRUCT DESIGNING
Eight CAR constructs (CAR1-CAR7 and CAR9) were in silico conceptualized and rationally synthesised at GenScript, USA. Structural elements of CAR (Table-1) consists of a signal peptide (CD8a/ TCRß), an anti-CD19 scFv, a hinge + a transmembrane domain (CD8a or CD8a / CH2CH3 or CD28), a co-stimulatory domain (4-1BB or CD28) and a signalling domain (CD3zeta).
Table-1: Structural Elements of CAR Construct
Sr No. Construct Signal peptide a-CD19 svFv
(source) Hinge + TM Co-stimulatory domain Signalling domain
1 CAR1 CD8a Murine CD8a 4-1BB CD3zeta
2 CAR2 CD8a Murine CD28 CD28 CD3zeta
3 CAR3 CD8a Human CD8a 4-1BB CD3zeta
4 CAR4 CD8a Human CD28 CD28 CD3zeta
5 CAR5 TCRß Blinatumomab CD8a 4-1BB CD3zeta
6 CAR6 TCRß Blinatumomab CD28 4-1BB CD3zeta
7 CAR7 TCRß Blinatumomab CH2CH3+ CD8a 4-1BB CD3zeta
8 CAR9 TCRß Xmab5774 CD8a 4-1BB CD3zeta

Vector map, gene cassette design and annotated sequence details of anti-CD19 CAR1-CAR7 and CAR9 constructs are shown in drawings (Figure 1 to Figure 8).

EXAMPLE 2: TRANSFORMATION, BANKING AND BULK DNA PREPARATION
• Transformation, clone selection and banking
The plasmid DNAs were transformed in an E. coli strain stbl3. Transformants were scored over LB-kanamycin. Plasmid DNAs were isolated from the transformants using Miniprep (QIAprep Spin Miniprep Kit) and restriction digestion analysis was performed to characterize the plasmid for the presence of gene of interest. Among several correct transformant, a high performing (high purity, yield as well as high content of covalently closed circular plasmid) clone was selected and final glycerol banks were prepared.

• Large scale DNA Preparation
A vial from the glycerol stock was grown over night at 37 °C for 16 to 18 hours in LB medium containing desired antibiotic (kanamycin). Bacterial pellet is subsequently harvested by pelleting the biomass via centrifugation at 4°C and large scale plasmid preparation was performed using Giga prep (Qiagen plasmid Giga Kits) as per manufacturer’s recommendation. Briefly, bacterial pellets were re-suspended in re-suspension buffer using a pipette followed by alkaline lysis, neutralization, column purification and concentration and purity determination at A280/A260.

EXAMPLE 3: GENERATION OF TRANSGENE PACKAGED LV PARTICLES AND TITER DETERMINATION

LV particles was produced using four plasmid based transient transfection of HEK293SF-3F6 suspension cell line. PEIMax was used as transfection reagent.
Chimeric antigen receptor lentivirus (CAR-LV) production in HEK293SF-3F6 cell line:
The lentivirus (LV) was packaged in HEK293SF-3F6 cell line applying 4 plasmid transfection with three helper plasmids namely pIntas-VSV-G, pIntas-Gag/Pol and pIntas-Rev and a transfer plasmid (pIntas-EF-1?-anti-CD19 CAR1- CAR9) in 1:3: 1:5 mass ratio), respectively. Briefly, HEK293SF-3F6 cells were grown in complete HyCell media supplemented with 4 mM of glutamine for transfection and generation of seed culture. Cells were seeded in disposable sterile shake flask with 0.5 × 106 cells/ml in 270 ml volume and incubated at 37°C, 8% CO2, and 125 rpm. 72 hours post seeding, the cell density was adjusted to 2.8-3.2 × 106 cells/ml and cells were transfected with plasmid mix formulated in PEI Max (1:1 ratio). Cells were incubated for next 72 hours at 37°C, 8% CO2, and 125 rpm. 72 hours post transfection, culture supernatant was harvested, filtered through a sterile 0.45 µm membrane filter and concentrated by ultracentrifugation. The obtained pallet was reconstituted in sterile HBSS media, aliquoted and stored at -80°C until future use.

Determination of functional titer of CAR-LV on adherent HEK 293FT cell line:
The functional titre determination was performed in a transduction assay by transducing HEK 293FT cell line with serially diluted CAR-LV. Briefly, HEK293FT cells were seeded in at the density of 50000 cells per well in 24 well plate in DMEM/F12 media supplemented with 10% FBS. Next day, cells were transduced using polybrene with varying dilutions of CAR-LV. Media change was given 24 hours post transduction and later, 72 hours after transduction, cells were stained for CAR positive and analyzed for CAR expression using flow cytometer. TU/ml was obtained in the range of 5.31E+05 to 4.31E+06 TU/ml for various CARs.

EXAMPLE 4: CELL LINE MAINTENANCE

• HEK293SF-3F6
HEK293SF-3F6 (Human Embryonic Kidney cells, suspension cells) cell line was propagated in their recommended HyCell media supplemented with 4 mM L-Glutamine in shake flask at 37°C, 8% CO2, and 125 rpm.

• HEK293FT
HEK293FT (Human Embryonic Kidney cells, adherent cells) cell line was propagated in their recommended media DMEM + 10% FBS in 75 cm2 cell culture treated flask (T75, Corning) at 37°C, 5% CO2.

• Raji-Luc/GFP
Raji-Luc/GFP (Mono) (Creative Biogene, USA) cell line was propagated in recommended media, RPMI-1640 supplemented with 10% FBS (fetal bovine serum) and 30 ug/ml Blasticidine in 25 cm2 cell culture non-treated flasks (for suspension cell line, Corning) at 37°C, 5% CO2. Cell density was maintained between 4 × 105 and 3 × 106 viable cells/ml.

• K562 mcherry/fluc
K562 mcherry/fluc cell line was propagated in recommended media, RPMI-1640 supplemented with 10% FBS (fetal bovine serum) and 1.5 µg/ml Puromycin in 25 cm2 cell culture non-treated flasks (for suspension cell line, Corning) at 37°C, 5% CO2. Cell density was maintained between 1 × 105 and 1 × 106 viable cells/ml

• Isolation and banking of pan T cells from buffy coat:
Residual buffy coats from peripheral blood of healthy donors were obtained and enrichment of CD3+ T cells was performed by negative selection by ready to use kit manufactures by Miltenvi Biotech (CD3 negative selection from buffy coat). Buffy Coat was diluted 1:2 to 1:3, stained with antibody cocktail containing antibodies conjugated to ferrous beads (magnetic). Upon applying magnetic force, all other cells of the buffy coat, except CD3, cells were bound to the magnet. Unbound CD3+ cells were collected for experimentation and cryopreservation. The immune cell composition (ICC) of the preparation was determined by flow cytometry and evaluated for the presence of NK cells, NKT cells, B cells, monocytes, CD4+ Thelper cells and CD8+ Teffector cell. Cell-preparation was formulated with cryostor CS10 (Sigma Aldrich), aliquoted and stored in liquid nitrogen for the future applications.

EXAMPLE 5: CAR T CELL GENERATION AND IN VITRO EVALUATION OF CAR EXPRESSION
Frozen aliquot of enriched CD3+ T cell preparation (n = 2 donors) was revived in TexMACS medium (Milteny) supplemented with 3% human AB serum (Sigma; H3667-100ML), 1350 IU/ml IL7 (Milteny), 450 IU/ml IL15 (Milteny) and activated with 1:100 dilution TransAct (Milteny). Next day cells were transduced at 0.3 MOI with CAR1- CAR7 and CAR9 separately and 48 hours later replenish with fresh medium with cytokines. Cells were expanded every alternate day and after next three days, CAR expression was determined using flow cytometry. CAR positivity for the various CARs (CAR1-CAR7 and CAR9) was observed in the range of 10.8% to 48%. Additionally, we observed two different phenotypes of CAR T cells during flow cytometric analysis namely; (i) dragged (CAR1, CAR3, CAR5 and CAR9) and (ii) compact (CAR2, CAR4, CAR6 and CAR7) for respective CARs (Figure 9).

EXAMPLE 6: IN VITRO POTENCY OF CAR T CELLS IN A CYTOTOXICITY ASSAYS
Potency of CAR T cells (n = 2 donors) were assessed for its CD19-specific cytotoxic potential by co-culturing CAR T cells [effector cells (E)] with CD19+ (Raji-Luc/GFP) and CD19- (K562-Fluc/cherry) expressing reporter cell lines [target cells (T)] at 3:1 and 6:1 effector (CAR-T cells) to target (Tumor cells) ratios (E : T). 24 hours later, co-culture was analyzed for the live cells expressing reporter fluorescent marker (GFP for Raji and mCherry for K562) using flow cytometer. Potency of CAR T cells expressing various anti-CD19 CAR (CAR1-CAR7 and CAR9) was evaluated at 20% CAR positivity i.e. effective E : T ratio 0.6 : 1.0 and 1.2 : 1.0. Two independent experiments in two technical replicates was performed from the CAR T cells generated from two different healthy donors (Figure 10). All the CAR T cells (CAR1-CAR7 and CAR9) were able to exhibit CD19-specific cytotoxicity (% cytotoxicity of Raji cells - % cytotoxicity of K562 cells) when co-cultured with CD19+ Raji cells and CAR3, CAR4, CAR6 and CAR9 exhibited equivalent cytotoxicity to CAR1 (Figure 10D and 10E). Further, at both the E: T ratios, we did not observed any significant (<10%; p-value > 0.05) non-CD19-specific cytotoxicity when CAR T cells were co-culture with CD19- K562 reporter cells (Figure 10B and 10C).

EXAMPLE 7: IN VITRO CHARACTERIZATION OF THE CAR T CELLS THROUGH PROCESS OF CAR T GENERATION
From the methods described in example 5 and 6, CAR1, CAR3, CAR6 and CAR9 were selected for further in vitro screening procedure. A full-fledged method for CAR T cell generation was used to fully characterize the CAR T cells in two different donor T cells. T/CAR T cells were evaluated for various characteristics like exhaustion markers, composition, persistence, CAR expression, cytotoxic potential and profile of cytokines released upon antigenic stimulation. CAR9 was excluded since T cells transduced with CAR9 LV showed higher PD1+TIM3+ markers which indicate exhaustive phenotype. On the other hand, levels of exhaustion markers for CAR1, CAR3 and CAR6 T cells were comparable to mock. Based on all aspects put together, CAR3 and CAR6 were selected for further evaluation in vivo (Figure 11A, 11B and 11C).

EXAMPLE 8: IN VIVO EVALUATION OF CYTOTOXIC POTENTIAL
To evaluate cytotoxic potential exerted by CAR T on CD19 positive tumor cells in vivo, NSG mice were used as the model animal. Briefly, NSG mice were infused with Tumor cell line (Raji) with Firefly Luciferase as the reporter gene. 4 days after tumor engraftment, 1 x 107 CAR T cells were infused through IV route. Bioluminescence as the marker of tumor progression/regression was used for evaluation of CAR3 and CAR6 cytotoxic potential in vivo. Based on the bioluminescent data, tumors regression was prominent in case of CAR6 as compared to CAR3. Thus, CAR6 was selected for further process development (Figure 12A, and 12B).
Table-2: Summary of analytical parameters of CAR T cells generated for POC study.
No. Parameter Test Performed Methodology Specification Mock CAR3 CAR6
1 Appearance Appearance Visual Report Results Cloudy Cloudy Cloudy
2 Purity CD3+ cell percentage Flow cytometry >85% 99.83% 99.64% 99.67%
3 CD3+ cell viability Flow cytometry >70% 98.67% 97.5% 97.48%
4 Residual B cells Flow cytometry <0.1% 0.015% 0.00% 0.005%
5 Residual Monocytes Flow cytometry <0.1% 0.00% 0.00% 0.00%
6 Identity % CAR+CD3+ cells Flow cytometry > 20% ND 60.4% 50.3%
7 Potency Cytotoxicity Flow cytometry >30% (E:T;3:1) ND 80% 95%
8 Safety Vector Copy Number Real time qPCR < 5 copies/cell ND 2.2 copies/cell 1.6 copies/cell
9 Bacterial Endotoxin/ml LAL < 0.5 EU <0.5 EU <0.5 EU <0.5 EU
10 Bioburden (d07) Culture No growth Negative Negative Negative
11 Sterility (d07 n d14) Culture No growth Negative Negative Negative
12 Mycoplasma Real time qPCR Negative Negative Negative Negative

Example 9: CRYOPRESERVATION
T/CAR T cells expanded ex vivo were cryopreserved using Stem Cell Banker (SCB) formulation (Make: Zenogen pharma). Cells were cryopreserved using SCB at a density of 50E+06 cells per ml per vial. Briefly, cells were counted, centrifuged and resuspended in ice cold SCB at a density of 50E+06 cells/ml. After overnight storage at -80 degree C, cells were transferred to liquid nitrogen for long term storage.

Table-3: CAR expression data of T/CAR T cells before and after cryopreservation.
Before cryopreservation After cryopreservation
CAR+ CAR+CD4+ CAR+CD8+ CAR+ CAR+CD4+ CAR+CD8+
CAR3 63.4 80.7 19 56.3 86.8 12.7
CAR6 53.5 85.5 14.1 49.4 90.2 8.34

Example 10: VECTOR COPY NUMBER (VCN) DETERMINATION
An aliquot of CAR T cells on the day of harvest is processed for gDNA isolation. Using primers/probe specific for WPRE is used for determination of number of viral genome integrated per cell of CAR positive population. Using qPCR and PTBP2 gene as the reference gene, number of integration is determined. As an additional proof, another sequence of viral genome (binder specific) was also used to validate the results obtained using WPRE/PTBP2 set of primers/probes. ,CLAIMS:We claim,
1. A Lentivirus (LV) vector comprising: nucleic acid sequence having SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor (CAR) against CD19.
2. The nucleic acid sequence encoding CAR against CD19 of claim 1, comprises:
(a) A signal peptide from CD8 alpha or TCR beta;
(b) CD19 antigen recognition sequence or fragment from monoclonal antibody;
(c) Extracellular spacer sequence or hinge region of CD8 alpha, CD28 or CH2-CH3 domain of IgG1;
(d) Transmembrane domain sequence of CD8 alpha or CD28; and
(e) One or multiple intracellular co-stimulatory 4-1BB and/or CD28; and
(f) CD3 zeta T-cell signalling domain.
3. A recombinant lentivirus (LV) vector comprising:
(a) CMV promoter; for viral RNA synthesis
(b) 5’ LTR;
(c) HIV-1 psi;
(d) RRE;
(e) cPPT/CTS;
(f) EF-1? for CAR expression;
(g) CAR1/CAR2/CAR3/CAR4/CAR5/CAR6/CAR7/CAR9;
(h) WPRE;
(i) 3’ LTR (Delta-U3); and
(j) SV40 poly (A) signal.
4. The recombinant lentivirus (LV) vector of claim 3, wherein the CAR is CAR6.
5. The Chimeric Antigen Receptor (CAR) according to any of the preceding claims is used for the treatment of CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia.
6. A method of treating CD19+ B cell malignancies, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and a subset of acute myeloid leukemia comprising administering a therapeutically effective amount of the recombinant lentivirus vector containing nucleic acid sequence having at least 85% identity to the nucleotide sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 encoding chimeric antigen receptor against CD19.