阅读说明：本技术 包含抗cd38和抗cd138嵌合抗原受体的t-细胞及其用途 (T-cells comprising anti-CD 38 and anti-CD 138 chimeric antigen receptors and uses thereof ) 是由 Z·伊萨哈 T·瓦克斯 A·格劳伯森莱温 M·拉维特斯诺博德金 于 2018-12-04 设计创作，主要内容包括：本发明提供了表达至少两种不同的嵌合抗原受体的T-细胞,其中所述CAR中的一者特异性结合到CD138,并且另一种CAR特异性结合到CD38。具体来说,本发明提供了表达两种不同CAR的T-细胞,其中一种CAR包含抗CD138 scFv,并且第二CAR包含抗CD138 scFv。此外,本发明提供了一种包含这些双重CAR T-细胞的药物组合物及其在癌症、特别是多发性骨髓瘤的治疗中的用途,以及制备这些细胞的方法。(The invention provides T-cells expressing at least two different chimeric antigen receptors, wherein one of the CARs specifically binds to CD138 and the other CAR specifically binds to CD 38. In particular, the invention provides T-cells expressing two different CARs, wherein one CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 138 scFv. Furthermore, the invention provides a pharmaceutical composition comprising these dual CAR T-cells and their use in the treatment of cancer, in particular multiple myeloma, as well as methods of making these cells.)

1. A T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein a first CAR comprises an antigen binding domain that specifically binds to CD138 and a second CAR comprises an antigen binding domain that specifically binds to CD 38.

2. The T-cell of claim 1, wherein the antigen-binding domain that specifically binds to CD138 and the antigen-binding domain that specifically binds to CD38 are each independently a single-chain variable fragment (represented as anti-CD 138scFv and anti-CD 38scFv, respectively).

3. The T-cell of claim 2, wherein the anti-CD 138scFv comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 or the like V_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: 14 or the like V_HA domain wherein said V of said anti-CD 138scFv_LAnd V_HThe domains are linked by a peptide linker, and wherein the analog has at least 70% identity to the original sequence.

4. The T-cell of claim 2 or 3, wherein the anti-CD 38scFv domain comprises a vh domain having the amino acid sequence of seq id NO: 15 or the like V_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: 16 or the like V_HA domain wherein V of said anti-CD 38scFv_LAnd V_HThe domains are linked by a peptide linker, and wherein the analog has at least 70% identity to the original sequence.

5. The T-cell of claim 3 or 4, wherein the peptide linker is a peptide having the amino acid sequence of SEQ ID NO: 17 or an analog thereof.

6. The T-cell of any one of claims 1-5, wherein at least one of the CARs comprises a co-stimulatory domain and another of the CARs comprises an activation domain.

7. The T-cell of claim 6, wherein the first CAR comprises an activation domain and does not comprise a co-stimulatory domain, and the second CAR comprises a co-stimulatory domain and does not comprise an activation domain.

8. The T-cell of claim 6, wherein the first CAR comprises a costimulatory domain and does not comprise an activation domain, and the second CAR comprises an activation domain and does not comprise a costimulatory domain.

9. The T-cell of claim 6, wherein at least one of the CARs comprises both an activation domain and a co-stimulatory domain.

10. The T-cell of any one of claims 6 to 9, wherein the co-stimulatory domain is selected from the group consisting of co-stimulatory domains of CD28, 4-1BB, OX40, igos, CD27, CD80, CD70, and the activation domain is selected from the group consisting of FcR γ and CD3- ζ activation domains.

11. The T-cell of any one of claims 6 to 10, wherein the activation domain is a polypeptide having the amino acid sequence of SEQ ID NO: 23 or an analog thereof, and the co-stimulatory domain is an FcR γ activation domain having the amino acid sequence of SEQ ID NO: 22 or analog thereof.

12. The T cell of claim 1, wherein the first CAR has the amino acid sequence of SEQ ID NO: 24 or an analog thereof, and the second CAR has the amino acid sequence SEQ ID NO: 25 or an analog thereof, and wherein the analog has at least 85% identity to the original sequence.

13. An engineered T-cell expressing two Chimeric Antigen Receptors (CARs), wherein a first CAR has an amino acid sequence set forth in SEQ id no: 24 or an analogue thereof, and the second CAR has the amino acid sequence of SEQ ID NO: 25 or an analog thereof, and wherein the analog has at least 85% identity to the original sequence.

14. The T-cell of claim 1, comprising at least one copy of one or more DNA constructs encoding the at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv.

15. The T-cell of claim 14, comprising at least one copy of a DNA construct encoding: (A) from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, and (B) from 5 'to 3' (v) a leader peptide, (vi) an anti-CD 38scFv domain, (vii) transmembrane domain II, and (viii) an activation domain, a costimulatory domain, or both; wherein (A) and (B) are separated by a self-cleaving peptide.

16. The T-cell of claim 16, wherein the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domain II, and (ix) an activation domain.

17. The T-cell of claim 15, wherein the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv, (iii) transmembrane domain II, (iv) an activation domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 138scFv domain, (viii) transmembrane domains I and (ix) a costimulatory domain.

18. The T-cell of any one of claims 15 to 17, wherein the self-cleaving peptide is selected from the group consisting of peptides having the amino acid sequence of SEQ ID NO: 26, an IRES peptide or analog thereof.

19. The T-cell of claim 18, wherein the self-cleaving peptide consists of the DNA sequence of SEQ ID NO: 27 or a variant thereof.

20. The T-cell of claim 14 comprising two different DNA constructs, wherein the first DNA construct comprises a DNA sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domains I and (iv) a costimulatory domain, an activation domain, or both, and the second DNA construct comprises a DNA sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv domain, (iii) transmembrane domains II and (iv) an activation domain, a costimulatory domain, or both).

21. The T-cell of any one of claims 14 to 20, wherein:

(i) the leader peptide has an amino acid sequence of SEQ ID NO: 20 or an analog thereof; and/or

(ii) The activation domain has the amino acid sequence SEQ ID NO: 23 or an analog thereof; and/or

(iii) The co-stimulatory domain has an amino acid sequence of SEQ ID NO: 22 or an analog thereof; and/or

(iv) The anti-CD 138scFv domain has the amino acid sequence SEQ ID NO: 18 or an analog thereof; and/or

(v) The anti-CD 38scFv domain has the amino acid sequence SEQ ID NO: 19 or an analogue thereof, or a pharmaceutically acceptable salt thereof,

wherein the analog has at least 85% sequence identity to the original sequence.

22. The T-cell of any one of claims 14 to 21, wherein:

(i) the leader peptide consists of SEQ ID NO: 39 or a variant thereof; and/or

(ii) The anti-CD 138scFv consists of SEQ ID NO: 28 or a variant thereof; and/or

(iii) The anti-CD 38scFv consists of SEQ ID NO: 29 or a variant thereof; and/or

(iv) The co-stimulatory domain consists of SEQ ID NO: 30 or a variant thereof; and/or

(v) The activation domain consists of SEQ ID NO: 31 or a variant thereof,

wherein the variant has at least 85% sequence identity to the original sequence.

23. The T-cell of claim 14, comprising a polypeptide having the DNA sequence of SEQ ID NO: 34 or a variant thereof.

24. The T-cell of claim 14, comprising a heavy chain variable region comprising the DNA sequence of SEQ ID NO: 32 or a variant thereof and a DNA construct comprising the DNA sequence of SEQ ID NO: 33 or a variant thereof.

25. The T-cell of any one of claims 1 to 24, wherein the T-cell is selected from the group consisting of a CD4+ T-cell and a CD8+ T-cell.

26. A DNA construct encoding:

(A) from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domain II, and (ix) a costimulatory domain, an activation domain, or both; or

(B) From 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv domain, (iii) transmembrane domain II, (iv) an activation domain, a costimulatory domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both.

27. The DNA construct of claim 26, wherein the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) a transmembrane domain II, and (ix) an activation domain.

28. The DNA construct of claim 26, wherein the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv, (iii) transmembrane domain II, (iv) an activation domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 138scFv domain, (viii) transmembrane domain I, and (ix) a costimulatory domain.

29. The DNA construct of any one of claims 26 to 28, wherein:

(i) the self-cleaving peptide has an amino acid sequence of SEQ ID NO: 26 or the like; and/or

(ii) The leader peptide has an amino acid sequence of SEQ ID NO: 20 or an analog thereof; and/or

(iii) The activation domain has the amino acid sequence SEQ ID NO: 23 or an analog thereof; and/or

(iv) The co-stimulatory domain has an amino acid sequence of SEQ ID NO: 22 or an analog thereof; and/or

(v) The anti-CD 138scFv domain has the amino acid sequence SEQ ID NO: 18 or an analog thereof; and/or

(vi) The anti-CD 38scFv domain has the amino acid sequence SEQ ID NO: 19 or an analogue thereof, or a pharmaceutically acceptable salt thereof,

wherein the analog has at least 85% sequence identity to the original sequence.

30. The DNA construct of any one of claims 26 to 29, wherein:

(i) the self-cleaving peptide consists of the DNA sequence SEQ ID NO: 27 or a variant thereof; and/or

(ii) The leader peptide consists of the DNA sequence SEQ ID NO: 39 or a variant thereof; and/or

(iii) The activation domain consists of the DNA sequence SEQ ID NO: 31 or a variant thereof; and/or

(iv) The co-stimulatory domain consists of a DNA sequence of SEQ ID NO: 30 or a variant thereof; and/or

(v) The anti-CD 138scFv domain consists of the DNA sequence SEQ ID NO: 28 or a variant thereof; and/or

(vi) The anti-CD 38scFv domain consists of the DNA sequence SEQ ID NO: 29 or a variant thereof,

wherein the variant has at least 85% sequence identity to the original sequence.

31. The DNA construct of claim 26, comprising the DNA sequence of SEQ ID NO: 32 and the DNA sequence SEQ ID NO: 33, or a variant thereof.

32. The DNA construct of claim 25, comprising the DNA sequence of SEQ ID NO: 34 or a variant thereof.

33. A vector comprising the DNA construct of any one of claims 26 to 32.

34. A cell comprising a DNA construct according to any one of claims 26 to 32 or a vector of claim 33.

35. The cell of claim 34, wherein the cell is a T-cell.

36. The cell of claim 35, wherein the T-cell is selected from the group consisting of a CD4+ T-cell and a CD8+ T-cell.

37. A pharmaceutical composition comprising a plurality of T-cells according to any one of claims 1 to 25, 35 or 36 and a pharmaceutically acceptable carrier.

38. The pharmaceutical composition of claim 37, comprising a pharmaceutically acceptable excipient.

39. The pharmaceutical composition of claim 37 or 38 for use in the treatment of cancer.

40. The pharmaceutical composition for use of claim 39, wherein the cancer is myeloma.

41. The pharmaceutical composition for use of claim 40, wherein myeloma is multiple myeloma.

42. A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the T-cell of any one of claims 1 to 25, 35 or 36.

43. A method for making a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein a first CAR comprises an anti-CD 138scFv and a second CAR comprises an anti-CD 38scFv, the method comprising transfecting a T-cell with a DNA construct according to any one of claims 26 to 32 or with a vector according to claim 33.

Technical Field

The present invention relates to T-cells expressing at least two Chimeric Antigen Receptors (CARs), wherein one of the CARs specifically binds to CD138 and the other CAR specifically binds to CD 38. The invention also relates to pharmaceutical compositions comprising said cells and their use in the treatment of cancer, in particular for the treatment of multiple myeloma.

Background

Chimeric antigen receptor T-cell (CAR-T) therapy is a newly developed adoptive anti-tumor therapy. Genetically modified T cells express a chimeric antigen receptor, which is typically composed of a signaling terminal domain (e.g., CD 3-zeta or gamma chain from the FcR), a transmembrane domain, and an extracellular single-chain variable fragment (scFv) derived from a monoclonal antibody that confers specificity to the receptor for a tumor-associated antigen on a target malignant cell. Upon binding of a tumor associated antigen by the chimeric antigen receptor, the chimeric antigen receptor expressed on a T cell (CAR T-cell) enhances an immune response that is cytotoxic to the malignant cell. In theory, CAR-T cells can specifically localize and eliminate tumor cells through interaction with Tumor Associated Antigens (TAAs) expressed on the surface of tumor cells.

Despite the rapid development in this field, the development of highly effective and safe CAR-T therapies still faces many challenges. Many known and numerous yet unidentified factors may contribute to the variability observed in different trials and clinical responses between individual patients. Factors that must be considered are, for example, the in vivo fate of the T-cells, the nature and safety of the tumor. To improve safety and efficacy, it was proposed to generate T-cells transduced with chimeric costimulatory receptors that provide suboptimal activation of CARs upon binding to one antigen and recognition of a second antigen (Kloss et al, 2013, Nature Biotechnology,13,71-75, WO2014/055668 and WO 2015/142314). An additional factor that is always one of the major obstacles in considering cancer therapy is the choice of target. True tumor-specific surface antigens are difficult to identify and the implementation of effective mechanisms to mitigate life-threatening and unexpected off-target toxicity is crucial.

Multiple Myeloma (MM) is an incurable Plasma Cell (PC) malignancy that occurs in the bone marrow and ultimately causes renal failure, immunosuppression with repeated infections, anemia, and skeletal lesions. Current therapies, including proteasome inhibitors and immunomodulators, have substantially improved outcomes in multiple myeloma patients. Unfortunately, most of these patients relapse, and treatment options are limited after exposure to these types of agents. Although CAR-T therapies can in principle overcome some of the problems of "classical" treatment of MM, a major obstacle in the development of CAR T-cell therapies is their significant on-target off-tissue toxicity. Only a few proteins, and indeed none of the cell surface antigens, are specifically expressed by malignant cells, resulting in unwanted targeting of healthy tissue. Therefore, finding antigens that are specifically expressed only on tumor cells has become a major goal in identifying CAR targets in MM. Several myeloma-related surface molecules have been identified as potential targets that can be targeted by CAR therapy. These targets include CD38, CD40, CD44, CD47, CD54, CD56, CD138, CD200, CD307, etc. (Atanockovic et al, British Journal of Haematology,2016,172, 685-698). However, none of these targets are exclusively present on cancer cells, and therefore significant off-target effects can be expected to occur when these targets are used in CAR-T cell therapy.

Chen et al (Leukemia 2017, doi:10.1038/leu.2017.302) indicated that targeting BCMA and CS1 on myeloma cells could potentially be an effective strategy to enhance responses to myeloma mass disease.

CD38 is a 45-kD type II transmembrane glycoprotein that binds to cell surface receptors on lipid rafts and regulates cytoplasmic Ca²⁺Flow and mediate signal transduction in lymphoid and myeloid lineage cells. CD38 is highly and uniformly expressed on myeloma cells and is expressed at relatively low levels on normal lymphoid and myeloid lineage cells as well as in certain tissues of non-hematopoietic origin, making it a potential target in myeloma therapy. A human IgG1 κ monoclonal antibody, daratumab (HuMax-CD38, GenMab), binds to a unique CD38 epitope. Preclinical studies have shown that darunavir induces target cell killing of CD 38-expressing tumor cells by a variety of mechanisms including complement-mediated and antibody-dependent cell-mediated cytotoxicity, antibody-dependent phagocytosis, apoptosis, and to a lesser extent inhibition of the enzymatic activity of CD 38. anti-CD 38 monoclonal antibodies are described, for example, in US 7,829,673.

CD138 is a surface protein that functions as an adhesion molecule that binds to the extracellular matrix molecules collagen and fibronectin. anti-CD 138 antibodies have been previously described, for example, in US9,221,914. Although CD138 is considered one of the most promising markers, in phase I/II studies using the immunoconjugate BT062 as a single agent, only 1 of 23 patients showed the targeted clinical response (Atanackovic et al). In addition, CD138 is expressed on many mature epithelial cells. Indeed, liver and skin toxicity was observed in those clinical trials, suggesting that significant side effects of CAR-T treatment against CD138 could be expected.

The other targets mentioned above also have their advantages and disadvantages, most of which are associated with high levels of side effects, which makes the selection of targets still one of the major obstacles to the development of safe and efficient CAR-T therapies. There is an unmet need in the rational development of other CAR T therapeutic systems that allow long-term safe treatment of multiple myeloma with fewer off-target side effects.

Disclosure of Invention

Now, in accordance with the present invention, T-cells genetically modified to express two separate different CARs capable of binding to two carefully selected different targets on multiple myeloma cells, namely CD38 and CD138, are disclosed, effectively treating the cancer and prolonging survival. Furthermore, the specific design of the CAR system, wherein one CAR carries only the activation domain and the second CAR carries only the co-stimulatory domain, allows to reduce the severity of the side effects associated with the "on-target off-tumor" binding of the T-cells. This design allows to obtain a highly effective treatment with reduced side effects. According to one aspect, the invention provides a T-cell genetically modified to express at least two different and separate Chimeric Antigen Receptors (CARs), wherein a first CAR comprises an antigen binding domain that specifically binds to CD138 and a second CAR comprises an antigen binding domain that specifically binds to CD 38. According to certain embodiments, the antigen binding domain that specifically binds to CD38 or CD138 is a single chain variable domain (scFv) of an anti-CD 38 and anti-CD 138 antibody, respectively. Thus, according to one embodiment, the invention provides a T-cell genetically modified to express two different and separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv.

According to certain embodiments, the anti-CD 138scFv comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 or the like V_LDomains and having the amino acid sequence SEQ ID NO: 14 or the like V_HA domain wherein said V of said anti-CD 138scFv_LAnd V_HThe domains are linked by a peptide linker and wherein the analogue has at least 70% identity to the parent (original) sequence.

According to other embodiments, the anti-CD 38scFv domain comprises a vh domain having the amino acid sequence of SEQ ID NO: 15 or the like V_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: 16 or the like V_HA domain wherein said V of anti-CD 38scFv_LAnd V_HThe domains are linked by a peptide linker, and wherein the analog has at least 70% identity to the original sequence.

According to certain embodiments, at least one of the CARs comprises a co-stimulatory domain and another of the CARs comprises an activation domain. According to one embodiment, the co-stimulatory domain is a co-stimulatory domain of CD28, 4-1BB, OX40, icaos, CD27, CD80, CD 70. According to other embodiments, the activation domain is selected from FcR γ and CD3- ζ.

According to some embodiments, the first CAR comprises a co-stimulatory domain and does not comprise an activation domain, and the second CAR comprises an activation domain and does not comprise a co-stimulatory domain. According to one embodiment, the activation domain has the amino acid sequence SEQ ID NO: 23 or an analog thereof, and the co-stimulatory domain has the amino acid sequence SEQ id no: 22 or the like.

According to some embodiments, the transmembrane domain of the first CAR has the sequence of SEQ ID NO: 35 or the like. According to other embodiments, the transmembrane domain of the second CAR has the sequence of SEQ ID NO: 36 or the like.

According to certain embodiments, the invention provides a CAR T-cell engineered to express two separate Chimeric Antigen Receptors (CARs), wherein the first CAR has the amino acid sequence of SEQ ID NO: 24 or an analog thereof, and the second CAR has the amino acid sequence of SEQ ID NO: 25 or an analog thereof.

According to one embodiment, the T-cells are CD4+ T-cells. According to another embodiment, the T-cells are CD8+ T-cells. According to any one of the above embodiments, the T-cell expresses a CAR of the invention.

According to another aspect, the invention provides a T-cell comprising at least one copy of one or more DNA constructs encoding two or more CARs of the invention. According to one embodiment, the T-cell comprises at least one copy of a DNA construct encoding: (A) from 5 'to 3' (I) a leader peptide, (ii) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both; and (B) from 5 'to 3' (v) a leader peptide, (vi) an anti-CD 38scFv domain, (vii) transmembrane domain II, and (viii) a costimulatory domain, an activation domain, or both; wherein (A) and (B) are separated by a self-cleaving peptide.

According to certain embodiments, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domain II, and (ix) an activation domain. According to other embodiments, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv, (iii) a transmembrane domain II, (iv) an activation domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 138scFv domain, (viii) transmembrane domain I, and (ix) a costimulatory domain. According to some embodiments, the self-cleaving peptide consists of the DNA sequence SEQ ID NO: 27 or a variant thereof.

Optionally, the T-cell comprises two different DNA constructs encoding a CAR of the invention, wherein the first DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domains I and (iv) a costimulatory domain, an activation domain, or both, and the second DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv domain, (iii) transmembrane domains II and (iv) a costimulatory domain, an activation domain, or both).

According to any one of the above embodiments, the anti-CD 138scFv consists of SEQ ID NO: 28 or a variant thereof. According to other embodiments, the anti-CD 38scFv consists of the DNA sequence of SEQ ID NO: 29 or a variant thereof. According to other embodiments, the co-stimulatory domain consists of the DNA sequence SEQ ID NO: 30 or a variant thereof. According to yet another embodiment, the activation domain consists of the DNA sequence SEQ ID NO: 31 or a variant thereof. According to some embodiments, the leader peptide consists of the DNA sequence of SEQ ID NO: 39 or a variant thereof. According to some embodiments, the transmembrane domain I consists of SEQ ID NO: 37 or a variant thereof. According to some embodiments, the transmembrane domain II consists of SEQ ID NO: 38 or a variant thereof.

According to one embodiment, the present invention provides a T-cell comprising a polypeptide having the DNA sequence of SEQ ID NO: 34 or a variant thereof.

According to other embodiments, the invention provides a CAR T-cell comprising at least one copy of each of two DNA constructs, wherein the first DNA construct comprises the DNA sequence of SEQ ID NO: 32 or a variant thereof, and said second DNA construct comprises the DNA sequence of SEQ ID NO: 33 or a variant thereof.

According to another aspect, the invention provides a DNA construct encoding at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an antigen binding domain that specifically binds to CD138 and the second CAR comprises an antigen binding domain that specifically binds to CD 38. According to one embodiment, the DNA construct encodes, from 5 'to 3': (i) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (iv) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domain II, and (ix) a costimulatory domain, an activation domain, or both. According to one embodiment, the present invention provides a DNA construct comprising the DNA sequence of SEQ ID NO: 32 and the DNA sequence SEQ ID NO: 33 or a variant thereof. According to another embodiment, the present invention provides a DNA construct comprising the DNA sequence of SEQ ID NO: 34 or a variant thereof.

According to another aspect, the present invention provides a vector comprising a DNA construct of the invention. According to one embodiment, the vector comprises a nucleic acid sequence comprising the DNA sequence of SEQ ID NO: 32 and the DNA sequence SEQ ID NO: 33 or a variant thereof. According to another embodiment, the vector comprises a nucleic acid sequence comprising the DNA sequence of SEQ ID NO: 34 or a variant thereof. According to certain embodiments, the vector is a viral vector.

According to yet another aspect, the invention provides a cell comprising a DNA construct or vector of the invention. According to one embodiment, the cell is a prokaryotic cell. According to another embodiment, the cell is a eukaryotic cell. According to some embodiments, the cell is a human cell. According to another embodiment, the cell is a T-cell such as a CD4+ or CD8+ T-cell.

According to another aspect, the invention provides a pharmaceutical composition comprising a plurality of CAR T-cells of the invention. According to one embodiment, the T-cells are genetically modified to express both CARs of the invention. According to one embodiment, the T-cell expresses a CAR of the invention. According to other embodiments, the T-cell comprises a DNA construct encoding two CARs of the invention or two or more different constructs encoding said two different CARs of the invention. According to certain embodiments, the pharmaceutical composition of the invention is for use in the treatment of cancer. According to one embodiment, the cancer is multiple myeloma. According to some embodiments, the T-cell is genetically modified to express a polypeptide having the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 25.

According to another aspect, the present invention provides a method of treating cancer, such as multiple myeloma, in a subject in need thereof, the method comprising administering an effective amount of a T-cell of the invention. According to another aspect, the invention provides a method for making a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 38scFv and the second CAR comprises an anti-CD 138scFv, the method comprising transfecting the T-cell with a DNA construct of the invention.

Drawings

Figure 1 shows the design scheme of a dual CAR DNA construct: v_LAnd V_HIs a portion of an scFv separated by a linker, T2A is a self-cleaving peptide, CD28 refers to a co-stimulatory element of CD28, and FcR γ is an activation domain.

Figure 2 shows the ability of T-cells transduced with different CAR constructs to interact with CAG cell lines expressing CD38 and CD138 tested by IFN- γ assay. The graph represents 4 replicate assays; the figure shows the secretion of IFN- γ, suggesting the presence of stimulation of different transduced T-cells as indicated in the figure.

Figure 3 shows stimulation of CAR T-cells by target cells expressing different levels of CD38 and CD 138: FIG. 3A-stimulation of different cell lines; FIG. 3 comparison of the activity of B-CAR T-cells on normal native tissue. The results are expressed as IFN-. gamma.concentration in pg/ml (ELISA).

Figure 4 shows that CAG-LUC cells are killed by dual CAR T cells. Luciferase-expressing CAG cells were incubated with transduced CAR T cells.

Figure 5 shows an image of anti-human CD138 antibody immunohistology of tissues from NSG mice transplanted with a human CAG multiple myeloma cell line compared to controls.

Figure 6 shows survival curves of mice injected with CAG multiple myeloma cells and treated with T-cells transduced with different CAR constructs.

Figure 7 shows hematoxylin and eosin (H & E) staining of tissues from surviving mice transplanted with CAG cell line and treated with SW1 or dual CARs.

FIG. 8 shows images of MM tumors expressing luciferase (CAG-Luc) in treated mice. Mice injected with MMCAG-Luc followed by different CARs (day 8) were monitored for therapeutic effect.

Figure 9 shows the toxicity of CD138 CAR T-cells on mouse skin (left panel mice-treated with CAR-CD 38T-cells), in contrast to the absence of this side effect when treated with dual CAR T-cells (right panel).

Detailed Description

According to one aspect, the invention provides a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein a first CAR comprises an antigen binding domain that specifically binds to human CD138 and a second CAR comprises an antigen binding domain that specifically binds to human CD 38.

As used herein, the term "T cell" refers to a class of lymphocytes produced or processed by the thymus, which, as is well known in the art, are involved in a variety of cell-mediated immune responses. The term encompasses T-cells transduced with nucleic acids, e.g., DNA or RNA, polypeptides, optionally using vectors. The T-cells of the invention are capable of expressing a CAR molecule encoded by the DNA or RNA used to transduce the T-cells by infection or electroporation.

The terms "chimeric antigen receptor" or "CAR" are used interchangeably herein and refer to an engineered receptor, i.e., a protein, that is expressed on a cell. Generally, a CAR comprises an extracellular domain (extracellular portion) including an antigen binding domain, a transmembrane domain, and an intracellular domain.

The extracellular domain comprises an antigen binding domain and optionally a spacer or hinge region.

The antigen binding domain of the CAR targets a particular antigen. The targeting region may comprise the full-length heavy chain of an antibody, a Fab fragment, or a single chain variable fragment (scFv) of an antibody. The antigen binding domain may be derived from the same or a different species as the species in which the CAR is to be used. In one embodiment, the antigen binding domain is an scFv.

The extracellular spacer or hinge region of the CAR is located between the antigen binding domain and the transmembrane domain. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, a helper protein, an artificial spacer sequence, or a combination thereof.

The term "transmembrane domain" refers to a region of a CAR that traverses or bridges the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein, an artificial hydrophobic sequence, or a combination thereof. According to certain embodiments, the term also encompasses a transmembrane domain together with an extracellular spacer or hinge region.

The term "specifically binds" or "specificity" with respect to an antibody, fragment thereof, or antigen binding domain of a CAR refers to an antigen binding domain that recognizes and binds a particular antigen, but does not substantially recognize or bind other molecules in a sample. The term implies that the antigen binding domain binds to its antigen with high affinity and to other antigens with low affinity. An antigen binding domain that specifically binds to an antigen from one species may also bind to the antigen from another species. This cross-species reactivity is not contradicted by the definition of the antigen-binding domain as specific.

The intracellular domain may be an intracellular domain of a T cell receptor or any other receptor (e.g., a member of the TNFR superfamily) or a portion thereof, such as an intracellular activation domain (e.g., an Immunoreceptor Tyrosine Activation Motif (ITAM) -containing T cell activation motif), an intracellular co-stimulatory domain, or both.

The terms "genetically modified T cell" and "CAR-T cell" of the invention are used interchangeably herein.

The terms "CD 38" and "human CD 38" are used interchangeably herein and refer to proteins designated human differentiation antigen cluster 38(CD38), ADP-ribosyl cyclase 1, cADPr hydrolase 1, cyclic ADP-ribosyl hydrolase 1, T10, and have the extension number EC 3.2.2.5. The term "anti-CD 38" or "α CD 38" refers to the antigen binding domain of an antibody that specifically binds to human CD 38. According to one embodiment, the antigen binding domain is an antigen binding domain of a CAR. According to another embodiment, the antigen binding domain is a scFv. According to other embodiments, the antigen binding domain binds to an epitope of human CD38, in particular an epitope of the extracellular domain of human CD 38.

The terms "CD 138" and "human CD 138" are used interchangeably herein and refer to a protein referred to as syndecan 1, SDC1, CD138, SDC or SYND1 and having the accession number P18827. The term "anti-CD 138" or "α CD 138" refers to the antigen binding domain of an antibody that specifically binds to human CD 138. According to one embodiment, the antigen binding domain is an antigen binding domain of a CAR. According to another embodiment, the antigen binding domain is a scFv. According to other embodiments, the antigen binding domain binds to an epitope of human CD138, in particular to an epitope of the extracellular domain of human CD 138.

According to certain embodiments, the antigen binding domain of the CAR of the invention is an scFv. According to one embodiment, the invention provides a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an scFv antigen binding domain that specifically binds to human CD138 (anti-CD 138scFv) and the second CAR comprises an scFv antigen binding domain that specifically binds to human CD38 (anti-CD 38 scFv).

According to one embodiment, the invention provides a T-cell genetically modified to express two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. As used herein, the term "first CAR" refers to a CAR that comprises an anti-CD 138 scFv. As used herein, the term "second CAR" refers to a CAR that comprises an anti-CD 38 scFv.

According to any of the above embodiments, the anti-CD 138 and anti-CD 38scFv binding domains comprise V_LAnd V_HA domain.

According to one embodiment, the anti-CD 138scFv comprises V_LAnd V_HA domain wherein said V_LThe domain comprises three Complementarity Determining Regions (CDRs) having the sequences SEQ ID NO: 1. 2 and 3 or consist thereof, and said V_HThe domain comprises three CDRs having the sequence SEQ ID NO: 4. 5 and 6 or consist thereof.

According to one embodiment, the anti-CD 38scFv comprises V_LAnd V_HA domain wherein said V_LThe domain comprises three Complementarity Determining Regions (CDRs) having the sequences SEQ ID NO: 7. 8 and 9 or consist thereof, and said V_HStructural domainsComprises three CDRs having the sequence SEQ ID NO: 10. 11 and 12 or consist thereof.

According to certain embodiments, the anti-CD 138scFv comprises 6 CDRs having the sequence of SEQ ID NO: 1. 2, 3, 4, 5 and 6, and the anti-CD 138scFv comprises 6 CDRs having the amino acid sequence of SEQ ID NO: 7. 8, 9, 10, 11 and 12, or a combination thereof.

According to any aspect and embodiment of the invention, the term "comprises SEQ ID NO: x, a peptide comprising the amino acid sequence shown in SEQ ID NO: x and "peptides having SEQ ID NO: peptide of X "is used interchangeably herein. The term "consists of SEQ ID NO: x, a peptide consisting of the amino acid sequence shown in SEQ ID NO: x and "SEQ ID NO: peptide of X "is used interchangeably herein.

According to any one of the above embodiments, the anti-CD 138scFv comprises a heavy chain variable region having the amino acid sequence of SEQ id no: 13 or the like V_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: 14 or the like V_HA domain wherein said V of said anti-CD 138scFv_LAnd V_HThe domains are linked by a peptide linker, and wherein the analog has at least 70% identity to the original sequence. According to one embodiment, the anti-CD 138scFv comprises a heavy chain variable region having the amino acid sequence of SEQ id no: v of 13_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: v of 14_HA domain. According to another embodiment, said V_LThe domain is SEQ ID NO: 13, or an analog thereof. According to other embodiments, said V_HThe domain is SEQ ID NO: 14, or an analog thereof. According to certain embodiments, the anti-CD 138scFv comprises a sequence that is SEQ ID NO: 13 of the analog V_LDomain and a sequence set forth as SEQ ID NO: 14 of the analogues V_HA domain.

According to any one of the above embodiments, the anti-CD 38scFv domain comprises a vh domain having the amino acid sequence of seq id NO: 15 or the like V_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: 16 or the like V_HA domain wherein said V of anti-CD 38scFv_LAnd V_HThe domains are linked by a peptide linker, and wherein the analog has at least 70% identity to the original sequence. According to one embodiment, the anti-CD 38scFv comprises a heavy chain variable region having the amino acid sequence of SEQ id no: v of 15_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: v of 16_HA domain. According to another embodiment, said V_LThe domain is SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof. According to another embodiment, said V_HThe domain is SEQ ID NO: 16. According to certain embodiments, the anti-CD 138scFv comprises a sequence that is SEQ ID NO: 15 of the analog V_LDomain and a sequence set forth as SEQ ID NO: 16 of the analogs V_HA domain.

The term "peptide linker" refers to any peptide capable of linking two variable domains, the length of which depends on the kind of variable domain to be linked. According to some embodiments, the peptide linker is a peptide having the amino acid sequence of SEQ ID NO: 17. According to another embodiment, the peptide linker is a peptide having SEQ ID NO: 17.

As described above, the scFv comprises a linker linked to V via a peptide linker_LV of the Domain_HA domain. According to some embodiments, said V_HAt V_LThe N-terminus of (1). According to another embodiment, said V_LAt V_HThe N-terminus of (1).

According to certain embodiments, the invention provides a T-cell genetically modified to express two CARs, wherein one CAR comprises an anti-CD 138scFv comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: v of 13_LA domain and a polypeptide having the amino acid sequence of SEQ id no: v of 14_HA domain, and the second CAR comprises an anti-CD 38scFv comprising a vh domain having SEQ ID NO: v of 15_LA domain and a polypeptide having the amino acid sequence of SEQ ID NO: v of 16_HA domain. According to certain embodiments, the V of the anti-CD 138scFv and/or anti-CD 38scFv_LAnd V_HThe domain is represented by a polypeptide having the amino acid sequence SEQ ID NO: 17 to a peptide linker.

The term "peptide" refers to a short chain of amino acid residues linked by peptide bonds, i.e. covalent bonds formed between the carboxyl group of one amino acid and the amino group of an adjacent amino acid. The term "peptide" refers to a short sequence of up to 50 amino acids. Chains of amino acid monomers longer than 50 amino acids are referred to as "polypeptides". These polypeptides, when having more than 50 amino acid residues, can also be classified as proteins, more specifically as low or medium molecular weight proteins.

The terms "peptide analog", "sequence analog", "similar sequence" and "SEQ ID NO: x "is used interchangeably herein and refers to an analog of a peptide having at least 70% sequence identity to the original peptide, wherein the analog retains the activity of the original peptide; x represents the number of the sequence. Thus, the terms "analog" and "active analog" may be used interchangeably. The term "analogue" refers to a peptide or protein that contains substitutions, rearrangements, deletions, additions and/or chemical modifications in the amino acid sequence of the parent (original) peptide or protein, respectively. According to certain embodiments, the peptide analog has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the original peptide. According to one embodiment, the analog has about 80% to about 99%, about 85% to about 98%, or about 90% to about 95% sequence identity to the original peptide. According to certain embodiments, the analogs of the invention comprise the sequence of the original peptide in which 1, 2, 3, 4, or 5 substitutions were made.

The substitution of amino acids may be conservative or non-conservative. Non-conservative substitutions encompass the replacement of one amino acid by any other amino acid.

According to certain embodiments, the term "analog" also encompasses the term "conservative analog".

Conservative substitutions of amino acids known to those skilled in the art are within the scope of the invention. Conservative amino acid substitutions involve the replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. The skilled artisan will recognize that individual substitutions are "conservatively modified analogs" where the change results in the substitution of an amino acid by a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. A typical example of conservative substitutions is provided below.

The following 6 groups each contain amino acids that are conservative substitutions for each other: (1) alanine (a), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) phenylalanine (F), tyrosine (Y), tryptophan (W). In other embodiments, conservative substitutions encompass substitutions using chemically similar unnatural amino acids.

Thus, in certain embodiments, the analog is a conservative analog of anti-CD 38 or anti-CD 138 scFv. According to certain embodiments, the conservative analogs of the invention comprise the sequence of the original scFv with 1, 2, 3, 4, or 5 conservative substitutions made therein. According to another embodiment, the analogue consists of the amino acid sequence of the original peptide in which 1, 2 or 3 conservative substitutions have been made. Thus, the analogs consist of the amino acid sequence of the original peptide with 1, 2, or 3 conservative substitutions.

According to some embodiments, the anti-CD 138scFv has the amino acid sequence of SEQ ID NO: 18. according to other embodiments, the anti-CD 138scFv consists of the amino acid sequence of SEQ ID NO: 18.

According to other embodiments, the anti-CD 38scFv has the amino acid sequence SEQ ID NO: 19. according to other embodiments, the anti-CD 38scFv consists of the amino acid sequence of SEQ ID NO: 19.

According to other embodiments, the anti-CD 138scFv has the amino acid sequence of SEQ ID NO: 18 and the anti-CD 38scFv has the amino acid sequence SEQ ID NO: 19. according to another embodiment, the anti-CD 138scFv consists of the amino acid sequence of SEQ ID NO: 18 and the anti-CD 38scFv consists of the amino acid sequence SEQ ID NO: 19.

According to any of the above embodiments, the extracellular domain of the CAR comprises a leader peptide. According to one embodiment, the leader peptide is located at the N-terminus of the scFv. The terms "leader peptide" and "signal peptide" are used interchangeably herein and refer to a peptide that translocates or facilitates the translocation of a target protein to the cell membrane.

According to one embodiment, the first CAR comprises a sequence N-terminal to the anti-CD 138scFv and comprising SEQ id no: 20. According to another embodiment, the polypeptide comprising SEQ ID NO: 20 is located within a polypeptide comprising SEQ id no: 18 or the N-terminus of an anti-CD 138scFv comprised thereof.

According to one embodiment, the second CAR comprises a sequence N-terminal to the anti-CD 38scFv and comprises SEQ ID NO: 20. According to another embodiment, the polypeptide comprising SEQ ID NO: 20 is located within a polypeptide comprising SEQ ID NO: 19 or the N-terminus of an anti-CD 38scFv comprised thereof.

According to any of the above embodiments, at least one of the CARs comprises a co-stimulatory domain and at least one further CAR comprises an activation domain.

According to some embodiments, the first CAR comprises a co-stimulatory domain and the second CAR comprises an activation domain. According to some embodiments, the first CAR comprises an activation domain and the second CAR comprises a co-stimulatory domain.

According to certain embodiments, the co-stimulatory domain is selected from the group consisting of co-stimulatory domains of CD28, 4-1BB, OX40, icaos, CD27, CD80, and CD 70. According to one embodiment, the co-stimulatory domain is the co-stimulatory domain of CD 28. According to another embodiment, the co-stimulatory domain has the amino acid sequence SEQ ID NO: 22. according to other embodiments, the co-stimulatory domain is SEQ ID NO: 22.

The terms "activation domain" and "activation element" are used interchangeably herein and refer to an intracellular signaling domain. According to certain embodiments, the activation domain is selected from FcR γ and CD3- ζ. According to one embodiment, the activation domain has the amino acid sequence SEQ ID NO: 23. according to other embodiments, the activation domain is SEQ id no: 23.

In accordance with the teachings of the present invention, it would be beneficial to separate the co-stimulatory domain from the activation domain in order to obtain a lower "on-target off-tumor" effect. Thus, according to one embodiment, one CAR comprises only the co-stimulatory domain and the other CAR comprises only the activation domain. Thus, in one embodiment, the first CAR comprising the anti-CD 138scFv comprises a costimulatory domain and no activation domain, and the second CAR comprising the anti-CD 38scFv comprises an activation domain and no costimulatory domain. According to another embodiment, the first CAR comprises an activation domain and does not comprise a co-stimulatory domain, and the second CAR comprises a co-stimulatory domain and does not comprise an activation domain.

According to other embodiments, at least one of the CARs comprises both an activation domain and a co-stimulatory domain.

According to another embodiment, the second CAR comprises both an activation domain and a co-stimulatory domain. According to such embodiments, the first CAR comprises a co-stimulatory domain and does not comprise an activation domain.

According to some embodiments, the activation domain has the amino acid sequence SEQ ID NO: 23 and the co-stimulatory domain has the amino acid sequence SEQ ID NO: 22. according to one embodiment, the activation domain is SEQ id no: 23. According to other embodiments, the co-stimulatory domain is SEQ ID NO: 22.

According to one embodiment, the first CAR comprises the amino acid sequence SEQ ID NO: 18 and SEQ ID NO: 22. according to another embodiment, the second CAR comprises the amino acid sequence SEQ ID NO: 19 and SEQ ID NO: 23. according to one embodiment, the invention provides an engineered T-cell comprising two CARs, wherein the first CAR comprises the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 22, and the second CAR comprises the amino acid sequence of SEQ id no: 19 and SEQ ID NO: 23.

according to any of the above embodiments, the CAR comprises a transmembrane domain (TM) and a hinge domain. According to the teachings of the present invention, when reference is made to a TM domain, it also includes a hinge domain, and the sequence of the transmembrane domain also includes the sequence of the hinge domain. According to one embodiment, the first CAR comprises a polypeptide having the amino acid sequence of SEQ id no: 35 TM domain I (TM-I). According to another embodiment, the second CAR comprises a polypeptide having the amino acid sequence of SEQ id no: 36 (TM-II).

According to certain embodiments, the invention provides a T-cell engineered to express two Chimeric Antigen Receptors (CARs), wherein the first CAR has the amino acid sequence of SEQ ID NO: 24, and the second CAR has the amino acid sequence shown in SEQ ID NO: 25. according to one embodiment, the first CAR has the amino acid sequence set forth as SEQ ID NO: 24. According to another embodiment, the second CAR has the amino acid sequence set forth as SEQ ID NO: 25, or a pharmaceutically acceptable salt thereof. According to other embodiments, the first CAR has a sequence similar to SEQ ID NO: 24, and the second CAR has an amino acid sequence similar to SEQ ID NO: 25. According to one embodiment, the invention provides a T-cell engineered to express two Chimeric Antigen Receptors (CARs), wherein the first CAR consists of the amino acid sequence of SEQ ID NO: 24 and the second CAR consists of the amino acid sequence SEQ ID NO: 25. According to some embodiments, the variant has at least 85% sequence identity to the original sequence. According to other embodiments, the variant has at least 90%, at least 95%, or at least 98% sequence identity to the original sequence.

According to any one of the preceding embodiments, the T cells are selected from the group consisting of CD4+ T-cells and CD8+ T-cells. Thus in one embodiment, the invention provides a CD4+ T-cell genetically modified to express two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to another embodiment, the invention provides a CD8+ T-cell genetically modified to express two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain such embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 22. according to another embodiment, the second CAR comprises the amino acid sequence SEQ ID NO: 19 and SEQ ID NO: 23. according to one embodiment, the first CAR comprises the amino acid sequence SEQ ID NO: 18 and SEQ ID NO: 22 and the second CAR comprises the amino acid sequence of seq id NO: 19 and SEQ ID NO: 23. according to one such embodiment, the first CAR has the amino acid sequence of SEQ id no: 24 or an analog thereof, and the second CAR has the amino acid sequence SEQ ID NO: 25 or an analog thereof. According to other embodiments, the invention provides a CD4+ and/or CD8+ T-cell genetically modified to express two different separate CARs, wherein the first CAR consists of the amino acid sequence of SEQ ID NO: 24 and the second CAR consists of the amino acid sequence SEQ ID NO: 25.

According to any of the above embodiments, the T-cell of the invention is capable of expressing two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain embodiments, the invention provides a T-cell expressing two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain embodiments, the T-cells are selected from CD4+ T-cells and CD8+ T-cells. Thus, in one embodiment, the invention provides a CD4+ T-cell expressing two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to another embodiment, the invention provides a CD8+ T-cell expressing two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain such embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 22. according to another embodiment, the second CAR comprises the amino acid sequence SEQ ID NO: 19 and SEQ ID NO: 23. according to one embodiment, the first CAR comprises the amino acid sequence SEQ ID NO: 18 and SEQ ID NO: 22, and the second CAR comprises the amino acid sequence of SEQ id no: 19 and SEQ ID NO: 23. according to one such embodiment, the first CAR has the amino acid sequence SEQ ID NO: 24 or an analog thereof, and the second CAR has the amino acid sequence SEQ ID NO: 25 or an analog thereof. According to other embodiments, the invention provides a CD4+ and/or CD8+ T-cell expressing two different separate CARs, wherein the first CAR consists of the amino acid sequence of SEQ ID NO: 24 and the second CAR consists of the amino acid sequence SEQ ID NO: 25.

According to another aspect, the invention provides a T-cell comprising at least one copy of one or more DNA constructs encoding at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an antigen binding domain that specifically binds to CD138 and the second CAR comprises an antigen binding domain that specifically binds to CD 38. According to one embodiment, the antigen binding domain of the CAR of the invention is an scFv. Thus, the T-cells comprise at least one copy of one or more DNA constructs encoding at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to one embodiment, the two CARs are encoded by one DNA construct. According to another embodiment, the two CARs are encoded by two different DNA constructs. According to one embodiment, the T-cell expresses or is capable of expressing a CAR of the invention.

As used herein, the term "DNA construct" refers to an artificially constructed nucleic acid segment. It may be an isolate or integrated into another DNA molecule. Thus, a "recombinant DNA construct" is produced by laboratory methods. The term "nucleic acid" encompasses single-or double-stranded DNA, RNA, and chemical modifications thereof. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein.

The CAR of the invention may be encoded by one DNA construct or by two or more DNA constructs.

According to certain embodiments, both CARs of the invention are encoded by one DNA construct. Thus in one embodiment, the invention provides a T-cell comprising at least one copy of a DNA construct encoding: (A) from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, and (B) from 5 'to 3' (v) a leader peptide, (vi) an anti-CD 38scFv domain, (vii) transmembrane domain II, and (viii) a costimulatory domain, an activation domain, or both; wherein (A) and (B) are separated by a self-cleaving peptide. According to one embodiment, the invention provides a T-cell comprising at least one copy of a DNA construct encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domains II and (ix) a costimulatory domain, an activation domain, or both.

According to one embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) a transmembrane domain II and (ix) an activation domain. According to another embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv, (iii) a transmembrane domain II, (iv) an activation domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 138scFv domain, (viii) transmembrane domain I and (ix) a costimulatory domain.

All definitions and embodiments used in the foregoing aspects, in particular those relating to CARs, their parts and domains, and to DNA constructs and T-cells, are encompassed and included herein.

According to one embodiment, the self-cleaving peptide is a peptide having the sequence of SEQ ID NO: 26 or an active analog thereof. According to another embodiment, the self-cleaving peptide is an IRES peptide or analog thereof. According to another embodiment, the self-cleaving peptide consists of the DNA sequence of SEQ ID NO: 27 or a variant thereof.

The terms "variant", "DNA variant", "sequence variant", "polynucleotide variant" and "SEQ ID NO: variants of X are used interchangeably herein and refer to DNA polynucleotides having at least 70% sequence identity to the original polynucleotide, where X is the number of the sequence. The variants may include mutations, such as deletions, additions or substitutions, such that the open reading frame is not altered by the mutations, and the polynucleotide encodes a peptide or protein having a structure and function substantially similar to the peptide or protein encoded by the original polynucleotide. According to certain embodiments, the variant is a conservative variant. As used herein, the term "conservative variant" refers to a variant in which a change in one or more nucleotides at a given codon position does not result in a change in the amino acid encoded at that position. Thus, the peptide or protein encoded by the conservative variant has 100% sequence identity to the peptide or protein encoded by the original polynucleotide. According to certain embodiments, the variant is a non-conservative variant of a peptide or protein that encodes a conservative analog of the peptide or protein encoded by the original polynucleotide. According to certain embodiments, the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the original polynucleotide.

According to certain embodiments, the CAR of the invention is encoded by two or more different DNA constructs. Thus in certain embodiments, the invention provides a T-cell comprising two different DNA constructs, wherein the first DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domains I (TM-I) and (iv) a costimulatory domain, an activation domain, or both, and the second DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv domain, (iii) transmembrane domains II (TM-II) and (iv) a costimulatory domain, an activation domain, or both). According to one embodiment, the first DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (ii) an anti-CD 138scFv, (iii) TM-I and (iv) a co-stimulatory domain. According to another embodiment, the second DNA construct comprises sequences encoding (i) a leader peptide, (II) an anti-CD 38scFv domain, (iii) a TM-II, and (iv) an activation domain. According to one embodiment, the first DNA construct comprises a sequence encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) TM-I and (iv) a costimulatory domain, and the second DNA construct comprises a sequence encoding (I) a leader peptide, (II) an anti-CD 38scFv domain, (iii) TM-II and (iv) an activation domain.

According to any of the above embodiments, either of the two CARs is encoded by one or two separate DNA constructs; the leader peptide has an amino acid sequence of SEQ ID NO: 20 or the like. According to another embodiment, the activation domain has the amino acid sequence SEQ ID NO: 23 or the like. According to other embodiments, the co-stimulatory domain has the amino acid sequence SEQ ID NO: 22 or the like. According to yet another embodiment, the anti-CD 138scFv domain has the amino acid sequence SEQ ID NO: 18 or the like. In one embodiment, the anti-CD 38scFv domain has the amino acid sequence of SEQ ID NO: 19 or the like. According to some embodiments, TM-I has the amino acid sequence of SEQ ID NO: 35. according to other embodiments, the TM-II has the amino acid sequence of SEQ ID NO: 36. according to some embodiments, the leader peptide consists of SEQ ID NO: 39 or a variant thereof; and/or the anti-CD 138scFv consists of SEQ ID NO: 28 or a variant thereof; and/or the anti-CD 38scFv is encoded by seq id NO: 29 or a variant thereof; and/or the co-stimulatory domain consists of SEQ ID NO: 30 or a variant thereof, and/or the activation domain is encoded by the DNA sequence shown in SEQ ID NO: 31 or a variant thereof. According to some embodiments, the transmembrane domain I consists of SEQ ID NO: 37 or a variant thereof. According to some embodiments, the transmembrane domain II consists of SEQ ID NO: 38 or a variant thereof. According to such embodiments, the analog or variant has at least 85% sequence identity to the original sequence.

According to other embodiments, the DNA constructs encoding both CARs comprise SEQ ID NO: 28. according to other embodiments, such DNA constructs comprise SEQ ID NO: 28, e.g., a conservative variant.

According to some embodiments, the two CARs are encoded by two different DNA constructs, and the first DNA construct comprises the DNA sequence of SEQ ID NO: 28. according to other embodiments, the first DNA construct comprises SEQ ID NO: 28, e.g., a conservative variant.

According to some embodiments, the DNA constructs encoding both CARs comprise SEQ ID NO: 29. according to other embodiments, such DNA constructs comprise SEQ ID NO: 29, such as a conservative variant.

According to some embodiments, the two CARs are encoded by two different DNA constructs, and the second DNA construct comprises the DNA sequences of SEQ ID NO: 29. according to other embodiments, the second DNA construct comprises SEQ ID NO: 29, such as a conservative variant.

According to some embodiments, the co-stimulatory domain consists of SEQ ID NO: 30 and the activation domain is encoded by the DNA sequence shown in SEQ ID NO: 31, or a pharmaceutically acceptable salt thereof.

According to one embodiment, both CARs are encoded by one DNA construct comprising, from 5 'to 3', the following DNA sequences: SEQ ID NO: 28. 30, 29 and 31.

According to some embodiments, the DNA construct comprises SEQ ID NO: 32 or a variant thereof. According to another embodiment, the DNA construct comprises SEQ ID NO: 33 or a variant thereof. According to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 32 and SEQ ID NO: 33. according to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 33 and SEQ ID NO: 32.

according to one embodiment, the invention provides a T-cell comprising a DNA construct encoding two CARs of the invention, wherein the DNA construct has the sequence of SEQ ID NO: 34. according to another embodiment, the DNA construct is SEQ ID NO: 34, such as a conservative variant. According to other embodiments, the variant has at least 90%, at least 95%, or at least 98% sequence identity to the original sequence.

According to some embodiments, the invention provides a T-cell comprising two DNA constructs encoding two CARs of the invention, wherein the first DNA construct comprises the DNA sequences of SEQ ID NO: 32 and the second DNA construct comprises the DNA sequence SEQ ID NO: 33. according to one embodiment, the first DNA construct has a DNA sequence that is SEQ ID NO: 32, e.g., conservative variants. According to one embodiment, said second DNA construct has a DNA sequence of seq id NO: 33, such as a conservative variant. According to other embodiments, the T-cell comprises two DNA constructs, wherein the first DNA construct comprises a DNA sequence that is SEQ ID NO: 32, and the second DNA construct has a DNA that is a variant of SEQ ID NO: 33, or a variant thereof. According to some embodiments, the variant has at least 85% sequence identity to the original sequence. According to other embodiments, the variant has at least 90%, at least 95%, or at least 98% sequence identity to the original sequence.

According to any one of the above embodiments, the T-cell comprising a DNA construct of the invention expresses or is capable of expressing the CAR encoded by the DNA construct.

As used herein, the word "expression" when referring to a DNA construct means the transcription and/or translation products of the construct. The expression level of a DNA molecule in a cell can be determined on the basis of the amount of the corresponding mRNA present in said cell or the amount of the protein encoded by the DNA produced by said cell. According to some embodiments, the expression is conditional expression.

According to another aspect, the invention provides a nucleic acid construct encoding two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an antigen binding domain that specifically binds to CD138 and the second CAR comprises an antigen binding domain that specifically binds to CD 38. According to one embodiment, the nucleic acid construct encodes two different separate chimeric antigen receptors, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to one embodiment, the nucleic acid construct is DNA. Thus in one embodiment the invention provides a DNA construct encoding two different separate chimeric antigen receptors wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv.

According to one embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) a transmembrane domain II, and (ix) a costimulatory domain, an activation domain, or both.

According to one embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) a transmembrane domain II and (ix) an activation domain. According to another embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 38scFv, (iii) a transmembrane domain II, (iv) an activation domain, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 138scFv domain, (viii) transmembrane domain I and (ix) a costimulatory domain. According to one embodiment, the leader peptide has the amino acid sequence SEQ ID NO: 20 or the like. According to another embodiment, the activation domain has the amino acid sequence SEQ ID NO: 23 or the like. According to other embodiments, the co-stimulatory domain has the amino acid sequence SEQ ID NO: 22 or the like. According to yet another embodiment, the anti-CD 138scFv domain has the amino acid sequence SEQ ID NO: 18 or the like. According to a certain embodiment, the anti-CD 38scFv domain has the amino acid sequence SEQ ID NO: 19 or the like. According to some embodiments, TM-I has the amino acid sequence of SEQ ID NO: 35. according to other embodiments, the TM-II has the amino acid sequence of SEQ ID NO: 36.

according to some embodiments, the leader peptide consists of SEQ ID NO: 39 or a variant thereof. According to other embodiments, the anti-CD 138scFv consists of SEQ ID NO: 28 or a variant thereof. According to certain embodiments, the anti-CD 38scFv consists of SEQ ID NO: 29 or a variant thereof. According to some embodiments, the co-stimulatory domain consists of SEQ ID NO: 30 or a variant thereof. According to yet another embodiment, the activation domain consists of SEQ ID NO: 31 or a variant thereof. According to some embodiments, the co-stimulatory domain consists of the DNA sequence SEQ ID NO: 30 and the activation domain is encoded by the DNA sequence SEQ ID NO: 31 are coded. According to some embodiments, the self-cleaving peptide consists of the DNA sequence SEQ ID NO: 27 or a variant thereof. According to some embodiments, the transmembrane domain I consists of SEQ ID NO: 37 or a variant thereof. According to some embodiments, the transmembrane domain II consists of SEQ ID NO: 38 or a variant thereof.

According to one embodiment, the DNA construct comprises, from 5 'to 3', the following DNA sequences: SEQ ID NO: 28. 30, 29 and 31.

According to some embodiments, the DNA construct comprises SEQ ID NO: 32 or a variant thereof. According to another embodiment, the DNA construct comprises SEQ ID NO: 33 or a variant thereof. According to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 32 and SEQ ID NO: 33. according to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 33 and SEQ ID NO: 32. according to some embodiments, the DNA construct comprises the sequence of SEQ ID NO: 32 and SEQ ID NO: 33 comprises a nucleotide sequence having the DNA sequence SEQ ID NO: 27.

According to some embodiments, the present invention provides a polypeptide comprising SEQ ID NO: 34. According to some embodiments, the present invention provides a polypeptide consisting of SEQ ID NO: 34. According to another embodiment, the DNA construct is SEQ ID NO: 34, such as a conservative variant.

According to some embodiments, the DNA construct is operably linked to a promoter. As used herein, the term "promoter" refers to a regulatory sequence that initiates transcription of a downstream nucleic acid. The term "promoter" refers to a DNA sequence within a larger DNA sequence that defines a site to which RNA polymerase can bind and initiate transcription. The promoter may include optional distal enhancer or repressing elements. The promoter may be homologous, i.e., occurring naturally to direct expression of the desired nucleic acid, or heterologous, i.e., occurring naturally to direct expression of a nucleic acid derived from a gene other than the desired nucleic acid. Promoters may be constitutive or inducible. Constitutive promoters are promoters that are active under most environmental and developmental conditions. Inducible promoters are promoters that are active under environmental or developmental regulation. A promoter may be derived in its entirety from a native gene, may comprise segments or fragments of a native gene, or may be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. It is also understood that the same promoter may be differentially expressed in different tissues and/or under different conditions.

According to another aspect, the present invention provides a vector comprising a nucleic acid construct of the invention. According to one embodiment, the DNA construct encodes two different separate chimeric antigen receptors, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to another embodiment, the vector comprises a DNA construct encoding from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) transmembrane domain II, and (ix) a costimulatory domain, an activation domain, or both. According to some embodiments, the DNA construct comprises, from 5 'to 3', the following DNA sequences: SEQ ID NO: 28. 30, 29 and 31. According to another embodiment, the DNA construct comprises SEQ ID NO: 32 and SEQ ID NO: 33. according to some embodiments, the DNA construct comprises a DNA sequence set forth in SEQ id no: 32 and SEQ ID NO: 33 comprises the sequence SEQ ID NO: 27. according to yet another embodiment, the present invention provides a vector comprising a nucleic acid sequence comprising SEQ ID NO: 34. According to other embodiments, the present invention provides a vector comprising a nucleic acid sequence consisting of SEQ ID NO: 34 in a DNA construct.

The terms "vector" and "expression vector" are used interchangeably herein and refer to any viral or non-viral vector, such as a plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), bacteriophage, binary vector in either double-stranded or single-stranded linear or circular form, or nucleic acid sequence capable of transforming a host cell and optionally capable of replicating in the host cell. The vector may be integrated into the genome of the cell, or may exist extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication). The vector may contain optional markers suitable for identifying the transformed cell, such as tetracycline resistance or ampicillin resistance. The cloning vector may or may not have the features necessary for its use as an expression vector.

According to other embodiments, the vector is a virus, e.g., a modified or engineered virus. Modifications of the virus may include mutations such as deletion or insertion mutations, gene deletions or gene inclusions. In particular, mutations can be made in one or more regions of the viral genome. Such mutations can be introduced into regions associated with internal structural proteins, replication or reverse transcription functions. Other examples of vector modifications are the deletion of certain genes that make up the native infectious vector, such as genes associated with the pathogenicity of the virus and/or its ability to replicate.

According to one embodiment, the vector is a viral vector. Any virus can be used by the methods disclosed herein. The virus may be a dsDNA virus (e.g., adenovirus, herpesvirus, poxvirus), a single-stranded "plus" sense DNA virus (e.g., parvovirus), a double-stranded RNA virus (e.g., reovirus), a single-stranded positive sense RNA virus (e.g., picornavirus, togavirus), a single-stranded "minus" sense RNA virus (e.g., orthomyxovirus, rhabdovirus), a single-stranded positive sense RNA virus (e.g., retrovirus) having a DNA intermediate, or a double-stranded retrovirus (e.g., hepadnavirus). In certain non-limiting embodiments of the invention, the virus is Poliovirus (PV), rhinovirus, influenza viruses including avian influenza (e.g., influenza A subtype H5N 1), Severe Acute Respiratory Syndrome (SARS) coronavirus, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), infectious bronchitis virus, Ebola virus, Marburg virus, dengue virus (flavivirus serotypes), west nile disease virus, epstein-barr virus (EBV), yellow fever virus, ebola (ebola virus), varicella (varicella-zoster virus), measles (paramyxovirus), mumps (paramyxovirus), rabies (rabies virus), human papilloma virus, carbopol sarcoma-associated herpes virus, herpes simplex virus (HSV type 1) or genital herpes (HSV type 2). According to certain embodiments, the vector is a virus selected from lentiviruses, adenoviruses, modified adenoviruses and retroviruses. In a particular embodiment, the vector is a lentivirus. According to any one of the above embodiments, the virus is a non-pathogenic virus or a modified virus lacking a pathogenic gene.

According to another aspect, the invention provides a cell comprising a DNA construct or vector of the invention. According to certain embodiments, the cell is a prokaryotic or eukaryotic cell. According to another embodiment, the cell is a non-human or human mammalian cell. According to some embodiments, the cell is a human cell. According to certain specific embodiments, the cell is a T-cell. According to one embodiment, the T-cells are selected from the group consisting of CD4+ T-cells and CD8+ T-cells.

According to another aspect, the present invention provides a pharmaceutical composition comprising a plurality of T-cells of the invention and a pharmaceutically acceptable carrier.

All definitions and embodiments used in the foregoing aspects, in particular those relating to CARs, their parts and domains, and to DNA constructs and T-cells, are encompassed and included herein.

The term "pharmaceutical composition" as used herein refers to a composition comprising the T-cells of the invention formulated together with one or more pharmaceutically acceptable carriers.

The formulation of the pharmaceutical composition may be adjusted depending on the application. In particular, the pharmaceutical compositions may be formulated so as to provide rapid, continuous or delayed release of the active ingredient upon administration to a mammal using methods known in the art. For example, the formulation may be any one selected from the group consisting of plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, eye ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluid ointments, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, spirits, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules. According to one embodiment, the composition is formulated as a liquid dosage form. According to another embodiment, the composition is formulated as a solution for injection.

As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coating agents, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds that provide supplemental, additional, or enhanced therapeutic functions.

The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities or compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human, as the case may be. For human administration, the formulations should meet sterility, pyrogenicity, overall safety and purity standards as required by governmental drug administration, such as the united states Food and Drug Administration (FDA) office of biologicals standards.

The compositions of the present invention may be administered by any known method. The term "administering" a composition or "administration" of a composition to a subject can be performed using one of a variety of different methods known to those of skill in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, for example, through a dermal catheter). The compound or agent may also suitably be introduced by replaceable or biodegradable polymeric devices or other devices such as patches and pumps or dosage forms for providing prolonged, slow or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time. In certain embodiments, the administration includes both direct administration (including self-administration) and indirect administration (including the act of prescribing a drug). For example, as used herein, a physician who instructs a patient to self-administer a drug or to administer the drug by others and/or to provide a patient with a prescription for a drug is administering the drug to the patient. According to one embodiment, the pharmaceutical composition is administered parenterally, i.e. not by oral administration. According to one embodiment, the pharmaceutical composition is administered systemically. According to another embodiment, the pharmaceutical composition is administered topically. According to one embodiment, the pharmaceutical composition is administered intravenously. According to another embodiment, the pharmaceutical composition is administered intramuscularly.

According to another embodiment, the pharmaceutical composition comprises a plurality of T-cells comprising the DNA construct of the invention. According to one embodiment, the T-cell comprises one construct encoding both CARs of the invention. According to another embodiment, the T-cell comprises two constructs, each encoding a separate CAR of the invention.

According to one embodiment, the pharmaceutical composition comprises a plurality of T-cells genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain embodiments, the T cells are selected from CD4+ T-cells, CD8+ T-cells, or a combination thereof. Thus, in one embodiment, the invention provides a pharmaceutical composition comprising a CD4+ T-cell genetically modified to express two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to another embodiment, the invention provides a pharmaceutical composition comprising a CD8+ T-cell genetically modified to express two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38 scFv. According to certain such embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 22. according to another embodiment, the second CAR comprises the amino acid sequence of SEQ id no: 19 and SEQ ID NO: 23. according to one embodiment, the first CAR comprises the amino acid sequence SEQ ID NO: 18 and SEQ ID NO: 22 and the second CAR comprises the amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 23. according to one such embodiment, the first CAR has the amino acid sequence SEQ ID NO: 24 or an analog thereof, and the second CAR has the amino acid sequence SEQ ID NO: 25 or an analog thereof. According to other embodiments, the invention provides a pharmaceutical composition comprising a CD4+ and/or CD8+ T-cell genetically modified to express two different separate CARs, wherein the first CAR consists of the amino acid sequence of SEQ ID NO: 24 and the second CAR consists of the amino acid sequence SEQ id no: 25. According to some embodiments, the T-cell is capable of expressing the CAR. According to another embodiment, the T-cell expresses the CAR.

According to one embodiment, the pharmaceutical composition comprises a T-cell comprising a DNA construct of the invention. According to another embodiment, the DNA construct encodes from 5 'to 3' (I) a leader peptide, (II) an anti-CD 138scFv, (iii) a transmembrane domain I, (iv) a costimulatory domain, an activation domain, or both, (v) a self-cleaving peptide, (vi) a leader peptide, (vii) an anti-CD 38scFv domain, (viii) a transmembrane domain II, and (ix) a costimulatory domain, an activation domain, or both. According to some embodiments, the DNA construct comprises, from 5 'to 3', the following DNA sequences: SEQ ID NO: 28. 30, 29 and 31. According to another embodiment, the DNA construct comprises SEQ ID NO: 32 and SEQ ID NO: 33. according to some embodiments, the DNA construct comprises the sequence of SEQ ID NO: 32 and SEQ ID NO: 33 comprises the sequence SEQ ID NO: 27. according to yet another embodiment, the present invention provides a vector comprising a nucleic acid sequence comprising SEQ ID NO: 34. According to other embodiments, the present invention provides a vector comprising a nucleic acid sequence consisting of SEQ ID NO: 34 in a DNA construct.

According to any one of the above embodiments, the pharmaceutical composition is for use in the treatment of cancer.

As used herein, the term "treating" a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, ameliorating or eliminating, substantially inhibiting, slowing or reversing the progression of cancer, substantially ameliorating or alleviating clinical or aesthetic symptoms of a disorder, substantially preventing the occurrence of a disease, clinical symptoms of a disorder, and protecting against harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder to be treated; (c) limiting the worsening of symptoms characteristic of the disorder to be treated; (d) limiting relapse of the disorder in a patient who has previously suffered from the disorder; and/or (e) limiting the recurrence of symptoms in a patient who was previously asymptomatic for the disorder.

According to one embodiment, the cancer is myeloma. According to another embodiment, the cancer is multiple myeloma.

The term "myeloma" refers to a cancer of the bone marrow. The term "multiple myeloma" refers to a cancer of plasma leukocytes.

Thus, in certain embodiments, the invention provides a pharmaceutical composition comprising a plurality of T-cells genetically modified to express two different separate CARs, wherein the first CAR comprises an anti-CD 138scFv and the second CAR comprises an anti-CD 38scFv, for use in the treatment of multiple myeloma. In certain embodiments, such pharmaceutical compositions are used to treat multiple myeloma. In one embodiment, the T cell is a CD4+ T-cell or a CD8+ T-cell genetically modified to express two different separate CARs. According to certain such embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 22. according to another embodiment, the second CAR comprises the amino acid sequence SEQ ID NO: 19 and SEQ ID NO: 23. according to one embodiment, the first CAR comprises the amino acid sequence of seq id NO: 18 and SEQ ID NO: 22 and the second CAR comprises the amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 23. according to one such embodiment, the first CAR has the amino acid sequence SEQ ID NO: 24 or an analog thereof, and the second CAR has the amino acid sequence SEQ ID NO: 25 or an analog thereof. According to other embodiments, the invention provides a CD4+ and/or CD8+ T-cell genetically modified to express two different separate CARs, wherein the first CAR consists of the amino acid sequence of SEQ ID NO: 24 and the second CAR consists of the amino acid sequence SEQ ID NO: 25. According to any one of the above embodiments, the T-cell expresses the two CARs.

According to any of the above aspects, the term treatment encompasses an increase in survival rate of at least 1.5, 2, 2.5 or 3 fold. According to another embodiment, the treatment comprises reducing side effects as compared to traditional treatment or as compared to treatment with T cells expressing one CAR.

According to certain embodiments, the treatment with the pharmaceutical composition has a much lower side effect rate.

According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof, said method comprising administering an effective amount of a T-cell of the invention. According to another embodiment, the method comprises administering a pharmaceutical composition comprising the T-cells of the invention. According to one embodiment, the cancer is myeloma. According to another embodiment, the cancer is multiple myeloma.

According to a further aspect, the invention provides the use of a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs) for the manufacture of a medicament for the treatment of cancer in a subject in need thereof, wherein the first CAR comprises an antigen binding domain that specifically binds to CD138 and the second CAR comprises an antigen binding domain that specifically binds to CD 38. According to one embodiment, the T-cell comprises a DNA construct of the invention. According to another embodiment, the T-cell comprises at least two DNA constructs that separately encode a CAR comprising an antigen binding domain that specifically binds to CD138 and a CAR comprising an antigen binding domain that specifically binds to CD 38.

According to another aspect, the present invention provides a method of making the T-cells of the invention. According to one embodiment, the invention provides a method of making a T-cell genetically modified to express at least two different separate Chimeric Antigen Receptors (CARs), wherein the first CAR comprises an antigen binding domain that specifically binds to CD138 and the second CAR comprises an antigen binding domain that specifically binds to CD38, the method comprising transfecting the T-cell with a DNA construct of the invention.

All definitions and embodiments used in the foregoing aspects, in particular those relating to CARs, their parts and domains, and to DNA constructs and T-cells, are encompassed and included herein.

The terms "transfection" or "transduction" are used interchangeably and are defined as the process of introducing a nucleic acid molecule into a cell. The nucleic acid is introduced into the cell using a non-viral or viral-based method. The nucleic acid molecule may be a gene sequence encoding a complete protein or a functional part thereof. Non-viral transfection methods include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, lipofection, nuclear transfection, sonoporation, transfection by heat shock, magnetic transfection and electroporation. In certain embodiments, the nucleic acid molecule is introduced into the cell using electroporation according to standard procedures well known in the art. For virus-based transfection methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral, and adeno-associated viral vectors. In certain embodiments, the nucleic acid molecule is introduced into the cell using a retroviral vector according to standard procedures well known in the art.

According to one embodiment, the T-cells are CD4+ T-cells. According to another embodiment, the T-cells are CD8+ cells.

According to one embodiment, the method comprises transfecting T cells with at least one DNA construct encoding two CARs, wherein the construct comprises, from 5 'to 3', the DNA sequences of SEQ ID NO: 28. 30, 29 and 31. According to some embodiments, the DNA construct comprises SEQ ID NO: 32 or a variant thereof. According to another embodiment, the DNA construct comprises seq id NO: 33 or a variant thereof. According to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 32 and SEQ ID NO: 33. according to another embodiment, the DNA construct comprises, from 5 'to 3', SEQ ID NO: 33 and SEQ id no: 32.

according to one embodiment, the DNA constructs of the invention encoding two CARs have the amino acid sequence of SEQ ID NO: 34. according to another embodiment, the DNA construct is SEQ ID NO: 34, such as a conservative variant.

According to one embodiment, the method comprises transfecting T cells with two DNA constructs each encoding a separate CAR of the invention, wherein the first DNA construct comprises the amino acid sequence of SEQ ID NO: 32, and the second DNA construct comprises the DNA sequence shown in SEQ ID NO: 33, or a DNA sequence shown in fig. 33. According to one embodiment, the first DNA construct has as a variant, for example, the nucleotide sequence of SEQ ID NO: 32, or a conservative variant thereof. According to one embodiment, the second DNA construct has as a variant, for example, the nucleotide sequence of SEQ ID NO: 33, or a conservative variant thereof. According to other embodiments, the T-cell comprises two DNA constructs, wherein the first DNA construct comprises a DNA sequence that is SEQ ID NO: 32, and the second DNA construct has a DNA that is a variant of SEQ ID NO: 33, or a variant thereof.

According to any one of the above embodiments, the transduction is performed using a viral vector selected from the group consisting of a retrovirus, an adenovirus, a lentivirus, and an adeno-associated viral vector.

According to certain embodiments, the vector may contain an optional marker suitable for identifying transformed cells.

The terms "comprising," including, "" having, "and" containing "are used interchangeably herein and have the meaning of" consisting at least in part of … …. When interpreting each statement in this specification that includes the term "comprising," features other than the one or more features prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same way. The terms "having" and "including" may also encompass, and be replaced by, the meanings of "consisting of … …" and "consisting essentially of … …". The term "consisting of … …" excludes any component, step, or procedure not specifically described or listed. The term "consisting essentially of … …" means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed composition or method.

Having now generally described the invention, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention.