Chimeric antigen receptor

Abstract

To provide a chimeric antigen receptor (CAR) which binds a low density target antigen and comprises a Fab antigen binding domain.SOLUTION: The invention relates to cells expressing such a CAR and their use in the treatment of disease. The present inventors have found that, for a CAR-T cell targeting a low-density target antigen, using a CAR with a Fab antigen binding domain results in more efficient CAR-mediated signalling. In an alternative embodiment of the first aspect of the invention, the Fab antigen binding domain may comprise a first binding domain which binds a first target antigen and a second binding domain which binds a second target antigen.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to chimeric antigen receptors (CARs) that bind to low density target antigens, such as B cell maturation antigen (BCMA). [Background technology]

[0002] 2. Background of the Invention Multiple myeloma Multiple myeloma (myeloma) is a bone marrow malignancy of plasma cells. Abnormal plasma cell clusters accumulate in the bone marrow where they interfere with the production of normal blood cells. Myeloma is the second most common hematological malignancy in the United States (after non-Hodgkin's lymphoma), comprising 13% of hematological malignancies and 1% of all cancers. The disease represents a significant burden given the health care expenditures caused by pathologic fractures, susceptibility to infection, renal failure, and subsequent bone marrow failure leading to death.

[0003] Unlike many lymphomas, myeloma is currently incurable. Standard chemotherapy agents used in lymphomas are largely ineffective against myeloma. Furthermore, loss of CD20 expression on plasma cells precludes the use of rituximab for this disease. Newer agents such as bortezamib and lenolidomide are partially effective but fail to induce sustained remission.

[0004] Therefore, there is a need for alternative drugs for the treatment of myeloma that are highly effective and have improved long-term efficacy.

[0005] Chimeric antigen receptors (CARs) Chimeric antigen receptors are proteins that combine the specificity of monoclonal antibodies (mAbs) with the effector functions of T cells in their usual form as type I transmembrane domain proteins with an antigen-recognizing amino terminus, a spacer, a transmembrane domain all connected with a compound endodomain that transmits T cell survival and activation signals.

[0006] The most common form of these molecules uses single chain variable fragments (scFv) derived from monoclonal antibodies to recognize target antigens. The scFv is fused to a signaling endodomain via a spacer and a transmembrane domain. Such molecules allow T cells to be activated in response to recognition of their target by the scFv. When T cells express such CARs, they recognize and kill target cells expressing the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for various cancer treatments. Carpenter et al (2013, Clin Cancer Res 19(8)2048-60) describe a CAR incorporating an scFv against B-cell maturation antigen (BCMA).

[0007] BCMA is a transmembrane protein preferentially expressed on mature lymphocytes (i.e., memory B cells, plasmablasts, and bone marrow plasma cells) and is expressed on multiple myeloma cells.

[0008] Carpenter et al. demonstrate that T cells transduced to express an anti-BCMA CAR can specifically kill myeloma cells derived from plasmacytomas in myeloma patients.

[0009] Although CAR approaches using anti-BCMA antibodies are promising, a particular problem in targeting this antigen is the particularly low density of BCMA on myeloma cells compared to, for example, CD19 on lymphoma cells. Although CAR-T cell-mediated treatments have been clinically successful against abundant target antigens such as CD19 or GD2, chimeric antigen receptors have been reported to be unable to signal in response to antigens at very low density.

[0010] For example, a CAR-T study targeting anaplastic lymphoma kinase (ALK) showed limited antitumor efficacy of CAR-T cells in two xenograft models of human neuroblastoma. Cytokine production was highly dependent on ALK target density, and target density of ALK on neuroblastoma cell lines was shown to be insufficient for maximal activation of CAR T cells (Walker et al. (2017) Mol. Ther. 25, 2189-2201).

[0011] Another study on the use of anti-CD22 CAR-T cells in the treatment of relapsed and / or refractory pre-B-cell acute lymphoblastic leukemia (B-ALL) found dose-dependent anti-leukemic activity, but some patients relapsed. Relapse was associated with a decrease in CD22 site density, which may have allowed CD22+ cells to avoid killing by CD22-CAR T cells (Fry et al. (2017) Nat. Med. doi:10.1038 / nm.4441).

[0012] CAR T cells undergo activation through stages that range from death, cytokine release, and proliferation. CAR T cells may kill target cells that have low density antigen, but cannot become fully activated.

[0013] Therefore, there is a need for alternative CAR T cell approaches that can kill target cells that express target antigens at low antigen density. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Carpenter et al(2013, Clin Cancer Res 19(8)2048-60 [Non-Patent Document 2] Walker et al.(2017)Mol.Ther.25,2189-2201 [Non-Patent Document 3] Fry et al. (2017) Nat.Med.doi:10.1038 / nm.4441 Summary of the Invention

[0015] Summary of the Invention The inventors found that for CAR-T cells targeting low-density target antigens, the use of CARs with Fab antigen-binding domains resulted in more efficient CAR-mediated signaling.

[0016] Thus, in a first aspect, the present invention provides a chimeric antigen receptor (CAR) that binds to a low density target antigen, wherein said CAR comprises a Fab antigen-binding domain.

[0017] The target antigen may be expressed at an average density of less than 1500 copies per target cell.

[0018] The target antigen may be selected from ROR1, Tyrp-1, TACI, ALK, and BCMA. For example, the target antigen may be BCMA.

[0019] In another embodiment of the first aspect of the invention, the Fab antigen binding domain may comprise a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen.

[0020] The first target antigen or the second target antigen may be BCMA.

[0021] In this embodiment, the FabCAR may comprise a cleavable linker between the first and second binding domains.

[0022] The linker may be cleavable using an agent such as an enzyme. For example, the linker may be cleavable using a matrix metalloproteinase (MMP). The agent may be administered to the subject before or after administration of the CAR-expressing cell of the present invention to cut out the first binding domain and expose the second binding domain. Alternatively, cleavage may occur naturally at a specific site if a cleavage agent (e.g., an enzyme) is expressed at the specific site. The site may be, for example, an inflammation site or a tumor site.

[0023] In a second aspect, the present invention provides a nucleic acid sequence encoding a CAR of the first aspect of the invention.

[0024] In a third aspect, the present invention provides a nucleic acid construct, encoding a CAR according to the first aspect of the invention, having the following general structure: VH-CH-spacer-TM-endo-coexpr-VL-CL; VL-CL-coexpr-VH-CH-spacer-TM-endo; VL-CL-spacer-TM-endo-coexpr-VH-CH; or VH-CH-coexpr-VL-CL-spacer-TM-endo; and Where: VH is a nucleic acid sequence encoding the heavy chain variable region; CH is a nucleic acid sequence encoding the heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides; VL is a nucleic acid sequence encoding the light chain variable region; CL provides a nucleic acid construct that is a nucleic acid sequence encoding a light chain constant region.

[0025] In another embodiment of the third aspect of the invention, the nucleic acid construct has the following general structure: VH1-L1-VH2-CH-spacer-TM-endo-coexpr-VL1-L2-VL2-CL; VL1-L1-VL2-CL-coexpr-VH1-L2-VH2-CH-spacer-TM-endo; VL1-L1-VL2-CL-spacer-TM-endo-coexpr-VH1-L2-VH2-CH; or VH1-L1-VH2-CH-coexpr-VL1-L2-VL2-CL-spacer-TM-endo and Where: VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first binding domain; L1 and L2 are cleavable linkers, which may be the same or different; VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second binding domain; CH is a nucleic acid sequence encoding the heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides; VL1 is a nucleic acid sequence encoding the light chain variable region of the first binding domain; VL2 is a nucleic acid sequence encoding the light chain variable region of the second binding domain; CL is a nucleic acid sequence encoding the light chain constant region.

[0026] The nucleic acid construct may also encode a second chimeric antigen receptor having a domain antibody (dAb), scFv, or Fab antigen-binding domain.

[0027] The second chimeric antigen receptor can bind one of the following antigens: CD19, FcRL5, and TACI.

[0028] In a fourth aspect, the present invention provides a vector comprising a nucleic acid sequence of the second aspect of the invention or a nucleic acid construct of the third aspect of the invention.

[0029] In a fifth aspect, the present invention provides a cell expressing a CAR of the first aspect of the invention.

[0030] The cell may express a first CAR of the first aspect of the invention as defined above, and a second chimeric antigen receptor.

[0031] The cells may also express constitutively active cytokine receptors.

[0032] In a sixth aspect, the present invention provides a method of producing a cell of the fifth aspect, the method comprising introducing into a cell ex vivo a nucleic acid sequence of the second aspect of the invention; a nucleic acid construct of the third aspect of the invention; or a vector of the fourth aspect of the invention.

[0033] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a plurality of the cells of the fifth aspect, together with a pharma- ceutically acceptable carrier, diluent or excipient.

[0034] In an eighth aspect, the present invention provides a method of treating cancer comprising administering to a subject a pharmaceutical composition of the seventh aspect of the claim.

[0035] The cancer can be multiple myeloma.

[0036] In a ninth aspect, the present invention provides a pharmaceutical composition according to the seventh aspect of the invention for use in the treatment of cancer.

[0037] In a tenth aspect, the present invention provides the use of a cell according to the fifth aspect of the invention in the manufacture of a pharmaceutical composition for treating cancer.

[0038] The present invention provides chimeric antigen receptors with improved CAR-mediated signaling delivery and target cell killing when targeting antigens expressed at low density on target cells, which are difficult to target using classical CARs because they form suboptimal T cell:target cell synapses (Figure 3).

[0039] Being able to target such antigens opens up entirely new possibilities for cancer treatment. Many potentially useful cancer target antigens are expressed at low density on target cells (such as ROR1, Tyrp-1, TACI, ALK, and BCMA). The present invention provides improved constructs for targeting these antigens, which can be used as single targets and, importantly, can be included in strategies for targeting multiple antigens to enhance the efficiency and safety of CAR-T cells.

[0040] Further Aspects The invention also provides aspects summarized in the following numbered paragraphs.

[0041] 1. A chimeric antigen receptor (CAR), comprising a Fab antigen binding domain that comprises a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen. 2. The CAR of paragraph 1, comprising a cleavable linker between the first and second binding domains. 3. The CAR of paragraph 2, wherein the linker is cleavable by a matrix metalloproteinase (MMP). 4. The CAR of any of the preceding paragraphs, wherein said first target antigen is expressed at a tumor site, such that when cells expressing said CAR are administered to a cancer patient, said cells home to the tumor site within said subject. 5. The CAR of any of the preceding paragraphs, wherein said second target antigen is expressed at the tumor site and also on one or more normal tissues. 6. A CAR according to any of the preceding paragraphs, wherein said first and / or second target antigens are selected from the group consisting of BCMA, ROR1, Tyrp-1, TACI, ALK, ErbB2, MUC1, CD33, CD123, PSMA, EpCAM, GD2, NCAM, folate binding protein, and MUC16. 7. The CAR of any of paragraphs 1 to 5, wherein said first target antigen / second target antigen is selected from one of the following antigen pairs: ErbB2 and MUC1; CD33 and CD123; PSMA and EpCAM; GD2 and NCAM; folate binding protein and MUC16. 8. The CAR of paragraph 7, wherein the first target antigen / second target antigen is one of the target antigen pairs shown in Table 2. 9. A nucleic acid sequence encoding a CAR according to any of the preceding paragraphs. 10. A nucleic acid construct encoding a CAR according to any of paragraphs 1 to 8, comprising the following general structure: VH1-L1-VH2-CH-spacer-TM-endo-coexpr-VL1-L2-VL2-CL; VL1-L1-VL2-CL-coexpr-VH1-L2-VH2-CH-spacer-TM-endo; VL1-L1-VL2-CL-spacer-TM-endo-coexpr-VH1-L2-VH2-CH; or VH1-L1-VH2-CH-coexpr-VL1-L2-VL2-CL-spacer-TM-endo and Where: VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first binding domain; L1 and L2 are cleavable linkers, which may be the same or different; VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second binding domain; CH is a nucleic acid sequence encoding the heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides; VL1 is a nucleic acid sequence encoding the light chain variable region of the first binding domain; VL2 is a nucleic acid sequence encoding the light chain variable region of the second binding domain; CL is a nucleic acid sequence encoding a light chain constant region, nucleic acid construct. 11. A vector comprising a nucleic acid sequence according to paragraph 9 or a nucleic acid construct according to paragraph 10. 12. A cell expressing a CAR according to any of paragraphs 1 to 8. 13. A method of producing a cell according to paragraph 12, comprising the step of introducing ex vivo into a cell a nucleic acid sequence according to paragraph 9; a nucleic acid construct according to paragraph 10; or a vector according to paragraph 11. 14. A pharmaceutical composition comprising a plurality of the cells of paragraph 12, together with a pharma- ceutically acceptable carrier, diluent, or excipient. 15. A method of treating cancer comprising administering to a subject the pharmaceutical composition according to paragraph 14. 16. A pharmaceutical composition according to paragraph 14 for use in the treatment of cancer. 17. Use of a cell according to paragraph 12 in the manufacture of a pharmaceutical composition for treating cancer. In certain embodiments, for example, the following are provided: (Item 1) A chimeric antigen receptor (CAR) that binds to a low density target antigen, wherein the CAR comprises a Fab antigen-binding domain. (Item 2) 2. The CAR of item 1, wherein the target antigen is expressed at an average density of less than 1500 copies per target cell. (Item 3) 3. The CAR of item 1 or 2, wherein the target antigen is selected from ROR1, Tyrp-1, TACI, ALK, and BCMA. (Item 4) The CAR of item 3, wherein the target antigen is BCMA. (Item 5) the Fab antigen-binding domain a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1 -GFIFSDYN (SEQ ID NO: 79) CDR2-IIYDGSST (SEQ ID NO: 80) CDR3-ATRPGPFAY (SEQ ID NO: 81); and b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences: CDR1-QSLLHSNGNTY (SEQ ID NO: 82) CDR2-LVS (SEQ ID NO: 83) CDR3-VHGTHAWT (SEQ ID NO: 84) 5. The CAR of item 4, comprising: (Item 6) The CAR of item 5, wherein the Fab antigen-binding domain comprises a VH domain having the sequence set forth as SEQ ID NO: 29; and a VL domain having the sequence set forth as SEQ ID NO: 30. (Item 7) The CAR of any of the preceding items, wherein the Fab antigen-binding domain comprises a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen. (Item 8) 8. The CAR of item 7, wherein the first target antigen or the second target antigen is BCMA. (Item 9) 9. The CAR of item 7 or 8, comprising a cleavable linker between the first and second binding domains. (Item 10) 10. The CAR of item 9, wherein the linker is cleavable using a matrix metalloproteinase (MMP). (Item 11) A nucleic acid sequence encoding a CAR according to any of the preceding items. (Item 12) A nucleic acid construct encoding a CAR according to any one of items 1 to 6, having the following general structure: VH-CH-spacer-TM-endo-coexpr-VL-CL; VL-CL-coexpr-VH-CH-spacer-TM-endo; VL-CL-spacer-TM-endo-coexpr-VH-CH; or VH-CH-coexpr-VL-CL-spacer-TM-endo; and Where: VH is a nucleic acid sequence encoding the heavy chain variable region; CH is a nucleic acid sequence encoding the heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides; VL is a nucleic acid sequence encoding the light chain variable region; CL is a nucleic acid sequence encoding a light chain constant region, nucleic acid construct. (Item 13) A nucleic acid construct encoding a CAR according to any one of items 7 to 10, having the following general structure: VH1-L1-VH2-CH-spacer-TM-endo-coexpr-VL1-L2-VL2-CL; VL1-L1-VL2-CL-coexpr-VH1-L2-VH2-CH-spacer-TM-endo; VL1-L1-VL2-CL-spacer-TM-endo-coexpr-VH1-L2-VH2-CH; or VH1-L1-VH2-CH-coexpr-VL1-L2-VL2-CL-spacer-TM-endo and Where: VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first binding domain; L1 and L2 are cleavable linkers, which may be the same or different; VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second binding domain; CH is a nucleic acid sequence encoding the heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides; VL1 is a nucleic acid sequence encoding the light chain variable region of the first binding domain; VL2 is a nucleic acid sequence encoding the light chain variable region of the second binding domain; CL is a nucleic acid sequence encoding a light chain constant region, nucleic acid construct. (Item 14) 14. The nucleic acid construct of item 12 or 13, which also encodes a second chimeric antigen receptor having a domain antibody (dAb), scFv, or Fab antigen-binding domain. (Item 15) 15. The nucleic acid construct of item 14, wherein the second chimeric antigen receptor binds to one of the following antigens: CD19, FcRL5, and TACI. (Item 16) A vector comprising the nucleic acid sequence according to item 9 or the nucleic acid construct according to any one of items 12 to 15. (Item 17) A cell expressing the CAR according to any one of items 1 to 10. (Item 18) A cell expressing a first CAR according to any of items 1 to 10 and a second chimeric antigen receptor as defined in item 14 or 15. (Item 19) 19. The cell of item 17 or 18, which also expresses a constitutively active cytokine receptor. (Item 20) 20. A method for producing the cell according to any one of Items 17 to 19, comprising the step of introducing ex vivo into a cell the nucleic acid sequence according to Item 11; the nucleic acid construct according to any one of Items 12 to 15; or the vector according to Item 16. (Item 21) 20. A pharmaceutical composition comprising a plurality of cells according to any of items 17 to 19, together with a pharma- ceutically acceptable carrier, diluent or excipient. (Item 22) A method for treating cancer, comprising administering to a subject the pharmaceutical composition according to item 21. 23. The method of claim 22, wherein the cancer is multiple myeloma. (Item 24) 22. The pharmaceutical composition according to item 21 for use in the treatment of cancer. (Item 25) 20. Use of a cell according to any of items 17 to 19 in the manufacture of a pharmaceutical composition for treating cancer. [Brief description of the drawings]

[0042] [Figure 1] BCMA expression in patients with multiple myeloma.

[0043] [Diagram 2] Different binding domain formats of chimeric antigen receptors. (a) Fab CAR format; (b) dAb CAR format; (c) scFv CAR format.

[0044] [Diagram 3] Schematic showing classical CAR-T cell interaction with target cells expressing target antigen at low density: (a) CAR T-cell membrane in the basal state; (b) CAR T-cell membrane forms an immune synapse with high-density target-expressing cells; (c) CAR T-cell membrane in response to target cells with low-density antigen.

[0045] [Figure 4] Schematic diagram showing a constitutively active chimeric cytokine receptor. The chimeric transmembrane protein comprises a dimerization domain and a cytokine receptor endodomain. The embodiment shown has a "Fab" type structure, as the dimerization domain comprises antibody-type heavy and light chain constant regions. Constitutive dimerization between these domains associates the IL2 receptor common gamma chain with either the IL-2 receptor beta chain or the IL-7 receptor alpha chain, leading to constitutive cytokine signaling.

[0046] [Diagram 5] Results of a killing assay comparing anti-BCMA CAR between scFv and FabCAR formats. CAR expressing cells were co-cultured with non-BCMA expressing target cells (SupT1 NT), low BCMA expressing target cells (SupT1BCMAlow), or ultra-low BCMA expressing target cells (JeKo-1) at a ratio of 1:4 (Figure 5A) or 1:8 (Figure 5B). A previously characterized anti-BCMA CAR (bb2121) was used as a positive control. After 24 hours, target cell killing was assayed by FACS and target cell numbers were normalized to cell numbers obtained with non-transfected T cells (NT).

[0047] [Figure 6] Results of a killing assay comparing anti-BCMA CAR between scFv and FabCAR formats. CAR expressing cells were co-cultured with non-BCMA expressing target cells (SupT1 NT), low BCMA expressing target cells (SupT1BCMAlow), or ultra-low BCMA expressing target cells (JeKo-1) at a ratio of 1:4 (Figure 6A) or 1:8 (Figure 6B). A previously characterized anti-BCMA CAR (bb2121) was used as a positive control. After 72 hours, target cell killing was assayed by FACS and target cell numbers were normalized to cell numbers obtained with non-transfected T cells (NT).

[0048] [Figure 7]IFNγ production by T cells expressing anti-BCMA CARs in either scFv or FabCAR format following co-culture with target cells. CAR expressing cells were co-cultured with non-BCMA expressing target cells (Figure 7A) or low BCMA expressing target cells (Figure 7B) at a ratio of 1:4 or 1:8. A previously characterized anti-BCMA CAR (bb2121) was used as a positive control. After 24 hours, IFNγ production was assayed by ELISA.

[0049] [Figure 8] CD4 and CD8 cell counts of T cells expressing anti-BCMA CARs in either scFv or FabCAR format after co-culture with target cells. CAR expressing cells were co-cultured with non-BCMA expressing target cells (SupT1 NT), low BCMA expressing target cells (SupT1BCMAlow), or ultra-low BCMA expressing target cells (JeKo-1), or MM.1s target cells at a 1:1 ratio for 96 hours. Whole cell counts of CD4 (Figure 8A) and CD8 (Figure 8B) T cells were obtained by gating on CD3 and the marker RQR8, then either CD8+ or CD8-.

[0050] [Figure 9] Schematic diagram showing a cleavable FabCAR. A cleavable FabCAR contains two antigen-binding domains: an external (membrane distal) binding domain 1 and an internal (membrane proximal) binding domain 2. Binding domains 1 and 2 are linked by a cleavable linker. Cleavage of the linker, for example by matrix metalloproteinases (MMPs), removes binding domain 1. In the absence of cleavage (when the FabCAR is intact), the internal binding domain is partially occluded by the presence of the external domain due to steric hindrance. The internal domain is activated when the heavy and light chains are cleaved.

[0051] [Figure 10]Schematic diagram showing the soluble antibody described in Example 7. The soluble antibody was designed with two variable domains, an external binding domain for BCMA (anti-BCMA D8) and an internal binding domain for CD19 (anti-CD19 CAT). An MMP-9 cleavable linker was included between the VH domain on one chain and the VL domain on the other chain. The VH-containing chain also contained the Fc portion of the antibody.

[0052] [Figure 11] Graph showing the cleavage rate of a soluble antibody (D8-MMP9-CAT19) over time after incubation in the presence of MMP-9. An equivalent antibody with a scrambled linker that does not contain an MMP-9 cleavage site (D8-scrambled-CAT19) was used as a negative control.

[0053] [Figure 12] Graph showing CD19 binding by soluble antibody (D8-MMP9-CAT19) or negative control with a scrambled linker (D8-scrambled-CAT19) either untreated or after treatment with MMP-9 using ELISA.

[0054] [Figure 13] Graph showing the binding kinetics of soluble antibodies to CD19 either untreated (D8-MMP9-CAT19) or after treatment with MMP-9 (D8-MMP9-CAT19 cleaved). CD19 monoclonal antibody (CAT19 IgG) was used as a positive control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Detailed Description Chimeric Antigen Receptor The present invention relates to a chimeric antigen receptor having a Fab antigen-binding domain.

[0056] Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins in which an extracellular antigen recognition domain (binder) is connected to an intracellular signaling domain (endodomain). The binder is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other formats that contain antibody-like antigen binding sites. A spacer domain is usually necessary to separate the binder from the membrane and allow for proper orientation. A common spacer domain used is the Fc of IgG1. Smaller spacers, for example, a stalk from CD8α or even just an IgG1 hinge, depending on the antigen, may be sufficient. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0057] Early CAR designs had endodomains derived from either the intracellular portion of the γ-chain of FcεR1 or CD3ζ. As a result, these first generation receptors were sufficient to transmit immunological signal 1 and trigger T cell killing of cognate target cells, but were unable to sufficiently activate the T cell so that it proliferates and survives. To overcome this limitation, composite endodomains were constructed as follows: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ results in a second generation receptor that can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal (i.e., immunological signal 2, which triggers T cell proliferation). Several receptors have also been described, including the TNF receptor family endodomains (such as the closely related OX40 and 41BB, which transmit survival signals). Here we describe even more potent third generation CARs with endodomains that can transmit activation, proliferation, and survival signals.

[0058] When a CAR binds to a target antigen, this binding transmits an activation signal to the T cell in which the CAR is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell to tumor cells expressing the targeted antigen.

[0059] Thus, a CAR typically comprises: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that comprises or is associated with a signaling domain.

[0060] A CAR can have the following general structure: Antigen-binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain).

[0061] Antigen-binding domain The antigen-binding domain is the part of the chimeric receptor that recognizes the antigen. In classical CARs, the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (see FIG. 2c). CARs with domain antibody (dAb) antigen-binding domains or VHH antigen-binding domains have also been produced (see FIG. 2b).

[0062] In the chimeric antigen receptor of the present invention, the antigen binding comprises, for example, the Fab fragment of a monoclonal antibody (see FIG. 2a). The FabCAR comprises two chains: a chain with an antibody-like light chain variable region (VL) and constant region (CL); and a chain with a heavy chain variable region (VH) and constant region (CH). One chain also comprises a transmembrane domain and an intracellular signaling domain. The association between CL and CH assembles the receptor.

[0063] The two chains of a Fab CAR may have the following general structure: VH-CH-spacer-transmembrane domain-intracellular signaling domain; and VL-CL or VL-CL-spacer-transmembrane domain-intracellular signaling domain; and VH-CH

[0064] For the Fab-type chimeric receptors described herein, the antigen-binding domain is composed of a VH from one polypeptide chain and a VL from the other polypeptide chain.

[0065] The polypeptide chain may include a linker between the VH / VL domain and the CH / CL domain, which may exhibit flexibility and serve to spatially separate the VH / VL domain from the CH / CL domain.

[0066] Flexible linkers may be composed of small, non-polar residues such as glycine, threonine, and serine. The linker may be (Gly4Ser) n (SEQ ID NO:78) linker, where n is the number of repeats. Each linker can be less than 50, 40, 30, 20, or 10 amino acids in length.

[0067] The present invention also provides a cleavable FabCAR comprising a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen. Such a cleavable Fab CAR is shown diagrammatically in FIG.

[0068] The cleavable Fab CAR may comprise a cleavable linker between the first and second binding domains.

[0069] The two chains of a cleavable Fab CAR may have the following general structure: VH1-VH2-CH-spacer-transmembrane domain-intracellular signaling domain; and VL1-VL2-CL or VL1-CL1-spacer-transmembrane domain-intracellular signaling domain; and VH2-CH2

[0070] In cleavable FabCAR, the membrane-proximal binding domain (binding domain 2 in FIG. 9) is less accessible in the uncleaved state due to steric hindrance by the first binding domain. Upon cleavage, binding domain 1 is removed, increasing the accessibility of binding domain 2, which then becomes activated in the presence of its target antigen.

[0071] The cleavable linker can be cleaved by an agent such as a small molecule or an enzyme such as a protease. An agent can be administered to a patient to cleave the linker and activate or enhance signal transduction through the second binding domain. Alternatively, the cleaving agent can be naturally expressed in vivo (e.g., at the site of inflammation or tumor growth / metastasis).

[0072] Matrix metalloproteinases (MMPs) are a group of enzymes responsible for the degradation of most extracellular matrix proteins during organogenesis and development. The expression and activity of MMPs in adult organs is normally low but is significantly increased in various pathological conditions, including inflammation and cancer.

[0073] MMPs are calcium-dependent, zinc-containing endopeptidases that share a common domain structure: a propeptide, a catalytic domain, and a hemopexin-like C-terminal domain linked to the catalytic domain by a flexible hinge region.

[0074] MMPs are present in almost all human cancers; they can be expressed by fibroblasts in the adjacent stroma, cancer-associated fibroblasts, and / or non-fibroblastic cancer cells. Thus, MMPs can affect the tumor environment by promoting angiogenesis, tumor growth, and metastasis. Thus, MMP expression is associated with tumor aggressiveness, disease stage, and patient prognosis.

[0075] Increased MMP expression correlates with increased cancer cell proliferation and increased tumor size. Nearly all members of the MMP family have been found to be dysregulated in human cancers, with MMP-1, -2, -7, -9, -13, and -14 being the most commonly affected.

[0076] In the cleavable FabCAR of the invention, the linker between the first and second binding domains can be cleaved by an MMP, in particular an MMP selected from MMP-1, -2, -7, -9, -13, and -14.

[0077] For example, various sequences cleavable by the following MMP enzymes are known: MMP-1: PLGLWA (SEQ ID NO:85) MMP-2: PAGLAG (SEQ ID NO:86) MMP-9: PLGLAG (SEQ ID NO:87)

[0078] The cleavable linker sequence of the cleavable FabCAR of the present invention may comprise one of the sequences shown as SEQ ID NOs: 85-87.

[0079] Constant Region Domain There are two types of light chains in humans: kappa (κ) and lambda (λ). The lambda class has four subtypes: λ1, λ2, λ3, and λ4. The light chain constant region of the Fab-type chimeric receptor can be derived from any of these light chain types.

[0080] The light chain constant domain of a chimeric receptor of the invention may have the sequence shown as SEQ ID NO:1, which is the kappa chain constant domain.

[0081] SEQ ID NO:1 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0082] There are five types of mammalian immunoglobulin heavy chains: gamma, delta, alpha, mu, and epsilon, which define the immunoglobulin classes IgG, IgD, IgA, IgM, and IgE, respectively. The heavy chains gamma, delta, and alpha have a constant domain made up of three tandem Ig domains and have a hinge that confers flexibility. The heavy chains mu and epsilon are made up of four domains.

[0083] The CH domain of a Fab-type chimeric receptor of the present invention may comprise the sequence shown as SEQ ID NO:2, which is derived from a gamma immunoglobulin heavy chain.

[0084] SEQ ID NO:2 STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0085] Spacer Classical CARs contain a spacer sequence that connects the antigen-binding domain with the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0086] In FabCAR (Figure 2a), the spacer allows the two polypeptide chains to dimerize, as in classical chimeric antigen receptors (Figure 2c) and dAb CARs (Figure 2b). The two polypeptide chains may, for example, contain one or more cysteine ​​residues suitable for forming disulfide bridge(s). An IgG1-derived hinge is suitable in this regard. A spacer based on an IgG1 hinge may have the sequence shown as SEQ ID NO:3.

[0087] SEQ ID NO:3 (human IgG1 hinge): AEPKSPDKTHTCPPCPKDPK

[0088] Alternatively, the hinge spacer may have the sequence shown as SEQ ID NO:4.

[0089] SEQ ID NO:4 (hinge spacer) EPKSCDKTHTCPPCP

[0090] Transmembrane domain The transmembrane domain is the part of the chimeric receptor that crosses the membrane. The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix that is composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor. The sequence and total length of the transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain can be used.

[0091] End Domain The endodomain is the signaling part of the chimeric receptor. The endodomain may be part of or associated with the intracellular domain of the chimeric receptor. After antigen recognition, the receptors cluster, native CD45 and CD148 are excluded from the synapse, and the signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta, which contains three ITAMs. It transmits activation signals to T cells after binding to antigen. CD3-zeta may not provide a fully qualified activation signal and further costimulatory signaling may be required. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used together with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0092] The endodomain may include: (i) an ITAM-containing endodomain (such as the endodomain from CD3 zeta); and / or (ii) a costimulatory domain (such as the endodomain from CD28 or ICOS); and / or (iii) a domain that transmits a survival signal (e.g., a TNF receptor family endodomain (such as OX-40, 4-1BB, CD27, or GITR)).

[0093] Several systems have been described in which the antigen recognition portion is on a separate molecule from the signaling portion (such as those described in WO015 / 150771; WO2016 / 124930, and WO2016 / 030691). Thus, the chimeric receptor of the present invention can comprise an antigen-binding component comprising an antigen-binding domain and a transmembrane domain, which can interact with a separate intracellular signaling component comprising a signaling domain. The vector of the present invention can express such a chimeric receptor signaling system comprising an antigen-binding component and an intracellular signaling component.

[0094] The chimeric receptor may contain a signal peptide so that when the signal peptide is expressed inside the cell, the nascent protein is targeted to the endoplasmic reticulum and then to the cell surface where it is expressed. The signal peptide may be at the amino terminus of the molecule.

[0095] target antigen A "target antigen" is the entity that is specifically recognized and bound by the antigen-binding domain of a chimeric receptor of the present invention.

[0096] The target antigen can be an antigen present on a cancer cell (e.g., a tumor-associated antigen).

[0097] The target antigen of the CAR may be expressed at a relatively low density on the target cell.

[0098] The cell of the present invention may be capable of killing target cells, such as cancer cells that express low density CAR target antigen.Examples of tumor-associated antigens that are known to be expressed at low density in certain cancers include, but are not limited to, ROR1 in CLL, Typr-1 in melanoma, BCMA and TACI in myeloma, and ALK in neuroblastoma.

[0099] Example 1 describes a study investigating the expression of BCMA on myeloma cells. A narrow range of BCMA copy numbers on the myeloma cell surface was found: 348.7 to 4268.4 BCMA copies per cell, with a mean of 1181 and a median of 1084.9 (Figure 1).

[0100] The average copy number of the target antigen of the CAR may be less than about 10,000; 5,000; 3,000; 2,000; 1,000; or 500 copies per target cell.

[0101] Copy number on the surface of cells, such as cancer cells, can be measured using standard techniques, such as PE Quantibrite beads as described in Example 1.

[0102] The target antigen of the CAR can be expressed by the target cell at an average copy number of 1500 copies per cell or less, or 1000 copies per cell or less.

[0103] The target antigen may be, for example, BCMA, ROR1, Tyrp-1, TACI, or ALK.

[0104] BCMA B-cell maturation target (BCMA; also known as TR17_HUMAN, TNFRSF17 (UniProt Q02223)) is a transmembrane protein expressed on mature lymphocytes (e.g., memory B cells, plasmablasts, and bone marrow plasma cells). BCMA is also expressed on myeloma cells. BCMA is a non-glycosylated type III transmembrane protein that is involved in B-cell maturation, growth, and survival.

[0105] The antigen binding domain of a CAR or TanCAR that binds to BCMA can be any domain capable of binding to BCMA. The VH and VL sequences of the 14 anti-BCMA antibodies are shown below, with the CDR sequences in bold and underlined.

[0106] [ka] [ka] [ka] [ka]

[0107] The antigen binding domain of the CAR that binds to BCMA may comprise the CDRs from any of the anti-BCMA Ab1 to Ab14, as described above.

[0108] The antigen binding domain of the CAR that binds to BCMA may comprise a VH and / or VL sequence from any of the anti-BCMA Ab1 to Ab14, as described above, or a variant thereof having at least 70, 80, 90, or 90% sequence identity, wherein said variants retain the ability to bind to BCMA.

[0109] The present invention also provides novel BCMA-binding antibodies, designated Ab1 to Ab14. The VH, VL and CDR sequences of Ab1 to Ab14 are shown above in or as SEQ ID NOs: 5 to 30.

[0110] The BCMA-binding antibody Ab1 shows particularly good efficacy in a CAR format, for example Ab1 in an scFv CAR format improved target cell killing, IL-2 release, and proliferation over a variety of equivalent CARs with different BCMA-binding domains (data not shown).

[0111] The invention also provides aspects summarized in the following numbered paragraphs.

[0112] 1. An antigen-binding domain comprising: a) a heavy chain variable region (VH) having a complementarity determining region (CDR) having the sequence set forth in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; and b) a light chain variable region (VL) having complementarity determining regions (CDRs) having the sequence set forth in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. 2. The antigen-binding domain of paragraph 1, comprising: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1-GFTFSDSY (SEQ ID NO: 68) CDR2-IYAGDGAT (SEQ ID NO: 69) CDR3-ARPLYTTAYYYVGGFAY (SEQ ID NO: 70); and b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences: CDR1 - QSLLSSGNQKNY (SEQ ID NO: 71) CDR2-WAS (SEQ ID NO: 72) CDR3-QQYYDTPLT (sequence number 73). 3. The antigen-binding domain of paragraph 1, comprising: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1 -GFIFSDYN (SEQ ID NO: 79) CDR2-IIYDGSST (SEQ ID NO: 80) CDR3-ATRPGPFAY (SEQ ID NO: 81); and b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences: CDR1-QSLLHSNGNTY (SEQ ID NO: 82) CDR2-LVS (SEQ ID NO: 83) CDR3 - VHGTHAWT (SEQ ID NO: 84). 4. An antigen-binding domain according to paragraph 1, comprising a VH domain having one of the sequences set out as SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; and a VL domain having one of the sequences set out as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. 5. An antigen-binding domain according to paragraph 3, comprising a VH domain having the sequence given as SEQ ID NO:5; and a VL domain having the sequence given as SEQ ID NO:6. 6. An antigen-binding domain according to paragraph 3, comprising a VH domain having the sequence given as SEQ ID NO:29; and a VL domain having the sequence given as SEQ ID NO:30. 7. An antibody comprising an antigen-binding domain according to any of the preceding paragraphs. 8. An antibody-drug conjugate (ADC) or a bispecific T cell derivative (BiTE) comprising an antibody according to paragraph 7. 9. A chimeric antigen receptor (CAR) comprising an antigen-binding domain according to any of paragraphs 1 to 6. 10. The CAR of paragraph 9, which is a FabCAR. 11. A nucleic acid sequence encoding an antigen-binding domain according to any of paragraphs 1 to 6, and an antibody according to paragraph 6, an ADC or BiTE according to paragraph 8, or a CAR according to paragraph 9 or 10. 12. A vector comprising a nucleic acid sequence according to paragraph 11. 13. A cell expressing the CAR of paragraph 9 or 10. 14. A method of producing a cell according to paragraph 13, comprising the step of introducing into a cell a CAR-encoding nucleic acid sequence according to paragraph 11. 15. A pharmaceutical composition comprising a plurality of the cells of paragraph 13, together with a pharma- ceutically acceptable carrier, diluent, or excipient. 16. A method of treating cancer comprising administering to a subject a pharmaceutical composition according to paragraph 15. 17. The method of paragraph 15, wherein the cancer is a B-cell leukemia or lymphoma. 18. The cells according to paragraph 13 for use in the treatment of cancer. 19. Use of a cell according to paragraph 13 in the manufacture of a pharmaceutical composition for treating cancer.

[0113] For example, the general features of the chimeric antigen receptors, nucleic acid sequences and constructs, vectors, cells, pharmaceutical compositions, and methods of making and using the cells described in the preceding and following sections also apply to the corresponding components described in the above paragraphs.

[0114] ROR1 Receptor tyrosine kinase-like orphan receptor-1 (ROR1), also known as neurotrophic tyrosine kinase and related receptor 1 (NTRKR1), is a receptor tyrosine kinase that regulates neurite outgrowth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors.

[0115] ROR1 has been shown to be expressed on ovarian cancer stem cells, where it appears to play a functional role in promoting migration / invasion or spheroid formation in vitro and tumor engraftment in immunodeficient mice. Treatment with a humanized mAb specific for ROR1 can inhibit the ability of ovarian cancer cells to migrate, form spheroids, or engraft in immunodeficient mice.

[0116] WO2017 / 072361 describes several ROR1-specific antibodies suitable for use in CARs.

[0117] TACI Transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) (UniProtKB:O14836) is a regulator in immune responses and, like BCMA, is preferentially expressed on mature lymphocytes such as CD27+ memory B cells (especially marginal zone B cells), bone marrow plasma cells, and myeloma cells.

[0118] Furthermore, TACI is expressed on macrophages and mediates their survival. TACI is a lymphocyte-specific member of the tumor necrosis factor (TNF) receptor superfamily (also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B)) and can be shed from the cell surface and circulate in its soluble form. In contrast to BCMA, TACI is conspicuously absent from germinal center B cells. TACI is known to function as a receptor for TNFSF13 / APRIL and TNFSF13B / BAFF, binding both ligands with high affinity. TACI inhibits B cell expansion and promotes plasma cell differentiation and survival.

[0119] TACI is a member of the tumor necrosis factor receptor (TNFR) superfamily and serves as a key regulator of B cell function. TACI binds two ligands (APRIL and BAFF) with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region. However, another form of TACI exists in which the N-terminal CRD is removed by alternative splicing. It has been shown that this shorter form is capable of ligand-induced cell signaling and that only the second CRD (TACI_d2) shows full affinity for both ligands (Hymowitz et al 2005 Am Soc. Biochem. And Mol. Biol. Inc 280(8)7218-7227).

[0120] The crystal structure of TACI_d2 has been solved, along with cocrystal structures of APRILTACI_d2 and the APRIL BCMA complex (Hymowitz et al 2005, supra).

[0121] The CRD of TACI, along with the CRDs of other TNFRs, shares a common sequence feature (the so-called DXL motif) consisting of a conserved six-residue sequence (Phe / Tyr / Trp)-Asp-Xaa-Leu-(Val / Thr)-(Arg / Gly) (SEQ ID NO: 74). This motif is required for binding of either APRIL or BAFF. The receptor motif binds to a hydrophobic pocket and interacts with two conserved arginine residues on the surface of BAFF.

[0122] The antigen-binding domain of a CAR or tanCAR that binds to TACI can be any domain that can bind to TACI. For example, the antigen-binding domain can include a TACI binder that can be derived from one of the commercially available anti-TACI antibodies listed in Table 1.

[0123] [Table 1]

[0124] The antigen binding domain may comprise one of the TACI binders from clones 2H6, 2G2, 1G6, or 4B11.

[0125] These TACI binders have the following sequences: 2H6 ScFv: EVQLQQSGPELVKPGASVRMSCKASGYTFTNYVMHWVKQKPGQGLEWIGYINPSNDDTKYTEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTHGDYYALDYWGQGTSVTVSSGGGGAGGGGSGGGGSDIVLTQSPASLAVSLGQSVTISCRASESVEYYGTSLMQWYQQKPGQAPKLLIYGASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR (SEQ ID NO: 31) CDR H1: NYVMH (SEQ ID NO: 32) CDR H2: YINPSNDDTKYTEKFKG (SEQ ID NO: 33) CDR H3: GTHGDYYALDY (SEQ ID NO: 34) CDR L1: RASESVEYYGTSLMQ (SEQ ID NO: 35) CDR L2: GASNVES (SEQ ID NO: 36) CDR L3: QQSRKVP (SEQ ID NO: 37)

[0126] 2G2 ScFv: QVTLKESGPGMLQPSQTLSLTCSFSGFSLSTFGMGVGWIRQPSGKGLEWLAHIWWDDAQYSNPALRSRLTISKDTSKNQVFLKIANVDTADTATYYCSRIHSYYSYDEGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTTVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYRTFGGGTKLEIKR (SEQ ID NO: 38) CDR H1: TGFMGVG (SEQ ID NO: 39) CDR H2: HIWWDDAQYSNPALRS (SEQ ID NO: 40) CDR H3: RIHSYYSYDEGFAY (SEQ ID NO: 41) CDR L1: KASQNVGTAVA (SEQ ID NO: 42) CDR L2: SASNRYT (SEQ ID NO: 43) CDR L3: QQYSSY (SEQ ID NO: 44)

[0127] 1G6 VH: QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVDWVRQSPGKGLEWLGIIWGGGRTNYNSAFKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCASGDRAADYWGQGTSVTVSS (SEQ ID NO: 45) CDR H1: SYGVD (SEQ ID NO: 47) CDR H2: IIWGGGRTNYNSAFKS (SEQ ID NO: 48) CDR H3: GDRAADY (SEQ ID NO: 49) VL:DIVMTQSQKFMSTTVGDRVTITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRYTGVPVRFTGSGSGTDFTLTINNMQSEDLADYFCQQYSSYPLTFGAGTKLELK (SEQ ID NO:46) CDR L1: KASQNVGTAVA (SEQ ID NO: 50) CDR L2: SASNRYT (SEQ ID NO:51) CDR L3: QQYSSYP (SEQ ID NO: 52)

[0128] 4B11 VH: EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYIHWVKQRPEQGLEWIGKIDPANGNSEYAPKFQGKATITADTSSNTAYLQLSSLTSEDTTIYYCTSGYGAYWGQGTTLTVSS (SEQ ID NO: 53) CDR H1: NTYIH (SEQ ID NO: 55) CDR H2: KIDPANGNSEYAPKFQG (SEQ ID NO: 56) CDR H3: GYGAY (SEQ ID NO: 57) VL: DIVLSQSPSSLAVSIGEKVTLSCKSSQSLLYSSNQKNYLAWFQQKPGQSLKLLIYWASTREFGVPDRFTGSGSGTDFTLTISSVKTEDLAVYYCQQYYTWTFGGGTKLEIK (SEQ ID NO: 54) CDR L1: KSSQSLLYSSNQKNYLA (SEQ ID NO:58) CDR L2: WASTREF (SEQ ID NO:59) CDR L3: QQYYTW (SEQ ID NO: 60)

[0129] The invention provides anti-TACI agents (such as antibodies, scFvs, CARs, or tanCARs) that comprise a TCI binding domain based on one of clones 2H6, 2G2, 1G6, or 4B11. The anti-TACI agents may comprise one or more CDRs having the sequences set forth as SEQ ID NOs: 32-37; 39-44; 47-52; 55-60. The agents may comprise one of the following six CDR groups: SEQ ID NOs: 32-37; SEQ ID NOs: 39-44; SEQ ID NOs: 47-52, and 55-60.

[0130] An anti-TACI agent may comprise, for example, in a Fab format, the VH and / or VL sequences shown in SEQ ID NO: 31 or 38 (wherein the VH is before the serine-glycine linker and the VL is after the serine-glycine linker). An anti-TACI agent may comprise, for example, in a Fab format, the VH sequence shown in SEQ ID NO: 45 or 53 and / or the VL sequence shown in SEQ ID NO: 46 or 54. An anti-TACI agent may comprise an scFv shown in SEQ ID NO: 31 or 38 or an scFv formed by linking SEQ ID NO: 45 to SEQ ID NO: 46 or linking SEQ ID NO: 53 to SEQ ID NO: 54.

[0131] For all of the above sequences, anti-TACI agents may include sequences having 80%, 85%, 90%, 95%, or 98% identity to a given sequence, where the variant sequence retains the ability to bind to TACI.

[0132] TYRP-1 Tyrp1 is a melanocyte-specific gene product involved in melanin synthesis. Mouse Tyrp1 has dihydroxyindole carboxylic acid oxidase activity, but its function in human melanocytes is poorly understood. In addition to its role in melanin synthesis, Tyrp1 is involved in stabilizing the tyrosinase protein and regulating its catalytic activity. Tyrp1 is also involved in maintaining melanosome structure and affects melanocyte proliferation and melanocyte cell death.

[0133] WO2009114585 describes several antibodies that bind to Tyrp-1, and anti-Tyrp-1 antibodies are commercially available from Sigma-Aldrich (such as SAB1406566 and HPA000937).

[0134] ALK Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that belongs to the insulin receptor superfamily. The protein contains an extracellular domain, a hydrophobic stretch corresponding to a single transmembrane region, and an intracellular kinase domain. Human ALK sequences are publicly available (e.g., GENBANK® accession numbers NP_004295 (protein), and NM_004304 (nucleic acid) and UniProt accession number Q9UM73).

[0135] WO2015069922 describes several scFv-type antigen-binding domains that bind to ALK.

[0136] Target antigen of cleavable Fab CAR As shown diagrammatically in FIG. 9, the Fab CAR of the present invention may have two antigen-binding domains.

[0137] In the cleavable FabCAR of the present invention, the first and / or second binding domain can bind to an antigen that is expressed at a relatively low level on target cells.For example, the aforementioned domain can bind to an antigen selected from ROR1, Typr-1, BCMA, TACI and ALK.In particular, the target antigen of the second (membrane proximal) binding domain can be an antigen that is expressed at a relatively low level on target cells.

[0138] CAR can target tumors using target antigen binding by the first (membrane distal) binding domain. When the linker is cleaved in the tumor, it activates through the binding of the second target antigen by the second binding domain, and tumor cells die. However, it should be noted that the binding of the first target antigen by the first binding domain can also induce cell death, but the killing efficiency may be reduced (compared to the killing induced by the target antigen binding via the second binding domain) due to inefficient synapse formation.

[0139] One advantage of cleavable FabCAR is that the internal binding domain (i.e., the second binding domain) is partially occluded within the intact molecule due to steric hindrance. This occlusion can improve safety when the second target antigen is expressed to some extent on one or more normal tissues, since the exposure of the second binding domain to its target antigen is reduced until the first binding domain is cleaved, which cannot occur until the CAR-expressing cell reaches the tumor.

[0140] Thus, the second antigen may be expressed at high levels on tumor cells, but at low levels on one of more normal tissues.

[0141] The target antigen of the first or second binding domain may be selected from one of the following: ErbB2, MUC1, CD33, CD123, PSMA, EpCAM, GD2, NCAM, folate binding protein, and MUC16.

[0142] In particular, the cleavable FabCAR of the invention may comprise binding domains directed against one of the following target antigen pairs: ErbB2 and MUC1; CD33 and CD123; PSMA and EpCAM; GD2 and NCAM; folate binding protein and MUC16. For these target antigen pairs, binding domain 1 and binding domain 2 may bind to either antigen of the target antigen pair, for example ErbB2 and MUC1, or may bind to either antigen of the binding domain. Binding domain 1 may bind to ErbB2 and binding domain 2 may bind to MUC1, or binding domain 2 may bind to ErbB2 and binding domain 1 may bind to MUC1.

[0143] In particular, the cleavable FabCAR of the present invention may comprise a binding domain directed against one of the target antigen pairs shown in Table 2.

[0144] [Table 2]

[0145] OR Gate

[0146] The CAR of the present invention may be used in combination with one or more other activating or suppressing chimeric antigen receptors.For example, the CAR of the present invention may be used in combination with one or more CARs in a "logic gate", where the CAR combination can detect a specific expression pattern of at least two target antigens when expressed by a cell such as a T cell.When the at least two target antigens are arbitrarily designated as antigen A and antigen B, the three possible options are as follows: "OR gate" - T cells trigger when either antigen A or antigen B is present on the target cell "AND gate" - T cells trigger when both antigens A and B are present on the target cell "AND NOT gate" - T cells trigger when antigen A is present alone on the target cell, but T cells do not trigger when antigens A and B are both present on the target cell

[0147] Engineered T cells expressing these CAR combinations can be tailored to be exquisitely specific for cancer cells based on the cancer cells' specific expression (or lack of expression) of two or more markers.

[0148] Such "logic gates" are described, for example, in WO2015 / 075469, WO2015 / 075470, and WO2015 / 075470.

[0149] An "OR gate" includes two or more stimulatory CARs, each directed to a distinct target antigen expressed by the target cell. The advantage of an OR gate is that the number of effectively targetable antigens on the target cell is increased, since the effectiveness is antigen A + antigen B. This is particularly important for antigens that are expressed at variable or low density on the target cell, since the level of a single antigen may be below the threshold required for CAR-T cells to effectively target it. OR gates also avoid the phenomenon of antigen escape. For example, some lymphomas and leukemias become CD19 negative after CD19 is targeted: if this phenomenon occurs, using an OR gate that targets CD19 in combination with another antigen provides a "backup" antigen.

[0150] The FabCAR of the present invention can be used in the OR gate in combination with a second CAR directed against a second target antigen expressed by the target cell.

[0151] For the anti-BCMA FabCAR, the OR gate may include a CAR against a second antigen expressed in B cells or plasma cells (such as CD19, TACI, or FcRL5).

[0152] The second CAR may have any suitable antigen binding domain (e.g., an scFv, a domain antibody (dAb), or a Fab-based binding domain).

[0153] The OR gate can be a double Fab CAR. In this regard, the cell can express two separate CARs with Fab-type antigen binding domains. The FabCARs can be, for example, against BCMA and TACI or BCMA and FcRL5.

[0154] The present invention also provides a triple OR gate comprising three CARs, at least one of which is a FabCAR according to the first aspect of the invention. The triple OR gate may, for example, comprise a BCMA Fab CAR, a CD19 CAR, and an FcRL5 CAR or a Fab CAR.

[0155] The second CAR may comprise a spacer that sterically separates the antigen-binding domain from the transmembrane domain and provides flexibility. A variety of sequences are commonly used as spacers in CARs (e.g., IgG1 Fc region, IgG1 hinge (above), or human CD8 stalk). The spacer may comprise, for example, a coiled-coil domain as described in WO2016 / 151315.

[0156] The second CAR comprises an activation endodomain. The second CAR can comprise, for example, an endodomain derived from CD3zeta. The second CAR can comprise one or more of the above-mentioned costimulatory domains. For example, the second CAR can comprise an endodomain derived from CD28, OX-40, or 4-1BB.

[0157] CD19 binder Several anti-CD19 antibodies have been previously described in the CAR format, such as fmc63, 4G7, SJ25C1, CAT19 (described in WO2016 / 139487) and CD19ALAb (described in WO2016 / 102965).

[0158] An anti-CD19 CAR for use in the double or triple OR gate of the present invention may comprise an antigen-binding domain, such as an scFv-type antigen-binding domain derived from one of these anti-CD19 antibodies.

[0159] The CD19 binding domain may comprise: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1-GYAFSSS (SEQ ID NO:61); CDR2-YPGDED (SEQ ID NO: 62) CDR3-SLLYGDYLDY (SEQ ID NO: 63); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO:64); CDR2-DTSKLAS (SEQ ID NO: 65) CDR3-QQWNINPLT (sequence number 66).

[0160] It may be possible to introduce one or more mutations (substitutions, additions, or deletions) into each CDR without negatively affecting CD19 binding activity. Each CDR may have, for example, one, two, or three amino acid mutations.

[0161] The CDRs can be in the form of a single chain variable fragment (scFv), which is a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody connected by a short linker peptide of 10 to about 25 amino acids. The orientation of the scFv can be VH-VL (i.e., the VH is at the amino terminal end of the CAR molecule, and the VL domain is linked to a spacer, followed by a transmembrane domain and an endodomain).

[0162] The CDRs can be grafted onto the framework of a human antibody or scFv. For example, the CAR of the present invention can comprise a CD19 binding domain consisting of or comprising one of the following sequences:

[0163] The anti-CD19 CAR may comprise the following VH sequence: SEQ ID NO:67 - VH sequence from mouse monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

[0164] The anti-CD19 CAR may comprise the following VL sequence: SEQ ID NO:75 - VL sequence from mouse monoclonal antibody QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0165] The anti-CD19 CAR may comprise the following scFv sequence: SEQ ID NO:76 - VH-VL scFv sequence from mouse monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSG GGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0166] The percent identity between two polypeptide sequences can be readily determined by programs such as BLAST, which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0167] FCRL5 Fc receptor-like protein 5 (FcRL5) is a member of the immunoglobulin receptor superfamily. Fc receptor-like family FcRL5 is a single-pass type I membrane protein that contains eight immunoglobulin-like C2-type domains. The mature protein is 106 kDa.

[0168] FCRL5 has a cytoplasmic tail with two inhibitory ITIM phosphorylation signaling motifs. FCRL5 inhibits B cell antigen receptor signaling by recruiting SHP1 upon costimulation of the B cell antigen receptor, thereby attenuating calcium influx and protein tyrosine phosphorylation. Costimulation of FCRL5 and the B cell antigen receptor promotes the proliferation and differentiation of naive B cells. FCRL5 is expressed on both mature B cells and plasma cells and is induced by EBV proteins. FCRL5 is overexpressed on malignant B cells from patients with hairy cell leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, and multiple myeloma.

[0169] Commercially available monoclonal antibodies against FcRL5 are known, such as CD307e (ThermoFisher) and REA391 (Miltenyi Biotec).

[0170] WO2016090337 describes several scFv-type antigen-binding domains that bind to FcRL5.

[0171] In the OR gate of the present invention, the anti-FcRL5 CAR may comprise a dAb, scFv, or Fab antigen-binding domain. The FcRL5 may comprise a Fab antigen-binding domain.

[0172] nucleic acid construct The present invention also provides nucleic acid constructs encoding the chimeric receptors of the present invention.

[0173] The nucleic acid construct encoding FabCAR (Figure 2a) may have the following structure: VH-CH-spacer-TM-endo-coexpr-VL-CL or VL-CL-spacer-TM-endo-coexpr-VH-CH Where: VH is a nucleic acid sequence encoding the heavy chain variable region; CH is a nucleic acid sequence encoding the heavy chain constant region spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding the endodomain; coexpr is a nucleic acid sequence enabling coexpression of the first and second polypeptides; VL is a nucleic acid sequence encoding the light chain variable region; CL is a nucleic acid sequence encoding the light chain constant region.

[0174] For both of the above constructions, the nucleic acid sequences encoding the two polypeptides can be in either order in the construct.

[0175] Contains two or more CARs, at least one of which Also provided is a nucleic acid construct encoding an OR gate, one of which is a FabCAR of the invention.

[0176] The nucleic acid construct encoding the double OR gate may have the following structure: VH-CH-spacer1-TM1-endo1-coexpr1-VL-CL-coexpr2-AgBD-spacer2-TM2-endo2; or VL-CL-spacer-TM1-endo1-coexpr1-VH-CH-coexpr2-AgBD-spacer2-TM2-endo2 Where: VH is a nucleic acid sequence encoding the heavy chain variable region of the first CAR; CH is a nucleic acid sequence encoding the heavy chain constant region of the first CAR; spacer1 is a nucleic acid sequence encoding the spacer of the first CAR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; endo1 is a nucleic acid sequence encoding the endodomain of the first CAR; coexpr1 and coexpr2 may be identical or different and are nucleic acid sequences capable of coexpression of the first CAR; and the first and second polypeptides of the first and second CARs; VL is a nucleic acid sequence encoding the light chain variable region of the first CAR; CL is a nucleic acid sequence encoding the light chain constant region of the first CAR; AgBD is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; endo2 is a nucleic acid sequence encoding the endodomain of the second CAR.

[0177] The antigen-binding domain of the second CAR can be, for example, an scFv or a dAb.

[0178] For both of the above constructs, the nucleic acid sequences encoding the two polypeptides of the first CAR; the nucleic acid sequences encoding the first and second CARs can be present in any order within the construct.

[0179] The nucleic acid construct encoding the double FabCAR OR gate may have the following structure: VH1-CH1-spacer1-TM1-endo1-coexpr1-VL1-CL1-coexpr2-VH2-CH2-spacer2-TM2-endo2-coexpr3-VL2-CL2; VH1-CH1-spacer1-TM1-endo1-coexpr1-VL1-CL1-coexpr2-VL2-CL2-spacer2-TM2-endo2-coexpr3-VH2-CH2; VL1-CL1-spacer1-TM1-endo1-coexpr1-VH1-CH1-coexpr2-VL2-CL2-spacer2-TM2-endo2-coexpr3-VH2-CH2; or VL1-CL1-spacer1-TM1-endo1-coexpr1-VH1-CH1-coexpr2-VH2-CH2-spacer2-TM2-endo2-coexpr3-VL2-CL2; Where: VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first CAR; CH1 is a nucleic acid sequence encoding the heavy chain constant region of the first CAR; spacer1 is a nucleic acid sequence encoding the spacer of the first CAR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; endo1 is a nucleic acid sequence encoding the endodomain of the first CAR; Coexpr1, coexpr2, and coexpr3 may be identical or different and are nucleic acid sequences allowing simultaneous expression of the first and second polypeptides of the first CAR; and the first and second polypeptides of the second CAR; VL2 is a nucleic acid sequence encoding the light chain variable region of the second CAR; CL2 is a nucleic acid sequence encoding the light chain constant region of the second CAR; VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second CAR; CH2 is a nucleic acid sequence encoding the heavy chain constant region of the second CAR; spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; endo2 is a nucleic acid sequence encoding the endodomain of the second CAR; VL2 is a nucleic acid sequence encoding the light chain variable region of the second CAR; CL2 is a nucleic acid sequence encoding the light chain constant region of the second CAR;

[0180] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0181] Those skilled in the art will understand that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art should understand that, using routine techniques, nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be substituted to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0182] The nucleic acid of the present invention may comprise DNA or RNA. The nucleic acid of the present invention may be single-stranded or double-stranded. The nucleic acid of the present invention may also be a polynucleotide that comprises synthetic or modified nucleotides within the nucleic acid of the present invention. Several different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that polynucleotides can be modified by any method available in the art for use as described herein. Such modifications can be performed to enhance the in vivo activity and extend the lifespan of the polynucleotide of interest.

[0183] The terms "variant," "homolog," or "derivative" in reference to a nucleotide sequence include any substitution, modification, alteration, replacement, deletion, or addition of one (or more) nucleic acid(s) from or to said sequence.

[0184] In the above construct, "coexpr" is a nucleic acid sequence that allows for the co-expression of two polypeptides as separate entities. Coexpr can be a sequence that encodes a cleavage site(s), such that the nucleic acid construct produces both polypeptides linked by the cleavage site(s). The cleavage site can be self-cleaving, such that when the polypeptides are produced, they are immediately cleaved into separate peptides without the need for any external cleavage activity.

[0185] The cleavage site may be any sequence that allows the two polypeptides to become separated.

[0186] The term "cleavage" is used herein for convenience, but the cleavage site may separate peptides into separate entities by mechanisms other than classical cleavage. For example, for the foot and mouth disease virus (FMDV) 2A autocleaving peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolytic activity by host cell proteinases, autoproteolysis, or translational effects (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as the cleavage site expresses the protein as a separate entity when placed between the nucleic acid sequences encoding the protein.

[0187] The cleavage site may be, for example, a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or may encode a self-cleaving peptide.

[0188] A "self-cleaving peptide" refers to a peptide that functions such that when proteins and polypeptides that include the self-cleaving peptide are produced, they are immediately "cleaved" or separated into individual and separate first and second polypeptides without the need for any external cleavage activity.

[0189] The self-cleaving peptide may be a 2A self-cleaving peptide from an aphthovirus or a cardiovirus. The primary 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In apthoviruses (such as foot and mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short stretch of about 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al. (2001) supra).

[0190] "2A-like" sequences have been found in picornaviruses other than aphthoviruses or cardioviruses, in "picornavirus-like" insect viruses, type C rotaviruses, and in repeated sequences within Trypanosoma spp and bacterial sequences (Donnelly et al., 2001, supra).

[0191] The cleavage site may include a 2A-like sequence (RAEGRGSLLTCGDVEENPGP) shown as SEQ ID NO:77.

[0192] Chimeric cytokine receptors A nucleic acid construct of the invention may also include one or more nucleic acid sequences encoding a chimeric cytokine receptor.

[0193] Chimeric cytokine receptors are described in WO2017 / 029512, which is incorporated herein by reference.

[0194] The chimeric cytokine receptor may include: an exodomain that binds to a ligand; and Cytokine receptor endodomains.

[0195] The ligand can be, for example, a tumor-secreted factor, a chemokine, or a cell surface antigen.

[0196] The chimeric cytokine receptor comprises two polypeptides: (i) a first polypeptide comprising: (a) a first antigen-binding domain that binds to a first epitope of the ligand; (b) a first chain of a cytokine receptor endodomain; and (ii) a second polypeptide comprising: (a) a second antigen-binding domain that binds to a second epitope of the ligand; (b) a second chain of the cytokine-receptor endodomain.

[0197] In another embodiment, the chimeric cytokine receptor may comprise a dimerization domain; and a cytokine receptor endodomain.

[0198] Dimerization may occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active, or it may occur only in the presence of a chemical inducer of dimerization (CID), in which case the transmembrane protein alone will produce cytokine-type signaling in the presence of the CID.

[0199] Suitable dimerization domains and CIDs are described in WO2015 / 150771, the contents of which are incorporated herein by reference.

[0200] For example, one dimerization domain may comprise the rapamycin binding domain of FK binding protein 12 (FKBP12) and the other may comprise the FKBP12-rapamycin binding (FRB) domain of mTOR; the CID may be rapamycin or a derivative thereof.

[0201] When the dimerization domain spontaneously heterodimerizes, this domain can be based on the dimerization domain of an antibody. In particular, the dimerization domain can include the dimerization portion of a heavy chain constant domain (CH) and a light chain constant domain (CL). The "dimerization portion" of the constant domain is the part of the sequence that forms interchain disulfide bonds.

[0202] For example, as shown diagrammatically in FIG. 4, a chimeric cytokine receptor may comprise the Fab portion of an antibody as an exodomain.

[0203] A chimeric cytokine receptor may comprise the following two polypeptides: (i) a first polypeptide comprising: (a) a first dimerization domain; and (b) a first chain of a cytokine receptor endodomain; and (ii) a second polypeptide comprising: (a) a second dimerization domain that dimerizes with the first dimerization domain; and (b) The second chain of the cytokine-receptor endodomain.

[0204] A chimeric cytokine receptor may comprise the following two polypeptides: (i) a first polypeptide comprising: (a) Heavy chain constant domain (CH) (b) a first chain of a cytokine receptor endodomain; and (ii) a second polypeptide comprising: (a) Light chain constant domain (CL) (b) The second chain of the cytokine-receptor endodomain.

[0205] A cytokine receptor endodomain can include, for example: (i) IL-2 receptor β chain endodomain (ii) the IL-7 receptor α chain endodomain; or (iii) the IL-15 receptor α chain endodomain; and / or (iv) The common γ-chain receptor endodomain.

[0206] A nucleic acid construct encoding a Fab-type constitutively active chimeric cytokine receptor may have the following general structure: CH-CRE1-coexpr-CL-CRE2; or CL-CRE2-coexpr-CH-CRE1 Where: CH is a nucleic acid sequence encoding the heavy chain constant domain of the first polypeptide; CRE1 is a nucleic acid sequence encoding the first chain of the cytokine receptor endodomain of the first polypeptide; coexpr encodes a sequence allowing for simultaneous expression of the first and second polypeptides; CL is a nucleic acid sequence encoding the heavy chain constant domain of the second polypeptide; CRE2 is a nucleic acid sequence encoding the second chain of the cytokine receptor endodomain of the second polypeptide.

[0207] The nucleic acid construct of the present invention can encode a FabCAR and a chimeric cytokine receptor.

[0208] vector The present invention also provides a vector or a kit of vectors comprising one or more nucleic acid sequences encoding the chimeric receptor of the present invention. Such vectors can be used to introduce the nucleic acid sequence(s) into a host cell such that the host cell expresses the CAR of the first aspect of the present invention.

[0209] Also provided are vectors comprising nucleic acid constructs of nucleic acid sequences encoding FabCAR and chimeric cytokine receptors.

[0210] The vector can be, for example, a plasmid or a viral vector (such as a retroviral or lentiviral vector), or a transposon-based vector or a synthetic mRNA.

[0211] The vector may be capable of transfecting or transducing a cell, such as a T cell or a NK cell.

[0212] cell The present invention provides a cell comprising the chimeric antigen receptor of the present invention. The cell may comprise two or more CARs (e.g., the cell may comprise the double OR gate or triple OR gate described above).

[0213] Also provided are cells co-expressing the FabCAR or OR gate of the invention and a chimeric cytokine receptor.

[0214] The cell may contain a nucleic acid or vector of the invention.

[0215] The cell can be a cytolytic immune cell (such as a T cell or a NK cell).

[0216] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of a T cell receptor (TCR) on the cell surface. There are different types of T cells, as summarized below.

[0217] T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes (TH1, TH2, TH3, TH17, Th9, or THF) that secrete different cytokines to facilitate different types of immune responses.

[0218] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and are also involved in graft rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0219] Memory T cells are a subset of antigen-specific T cells that persist for extended periods of time after recovery from an infection. Memory T cells rapidly expand into large numbers of effector T cells upon reexposure to their cognate antigen, thus providing the immune system with a "memory" of past infection. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0220] Regulatory T cells (Treg cells), previously known as suppressor T cells, are essential for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of the immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0221] Two main classes of CD4+ Treg cells have been described: intrinsic and adaptive Treg cells.

[0222] Endogenous Treg cells (also known as CD4+CD25+FoxP3+Treg cells) arise in the thymus and are involved in the interaction of developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that are activated with TSLP. Endogenous Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutation of the FOXP3 gene can block the development of regulatory T cells and cause the fatal autoimmune disease IPEX.

[0223] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during normal immune responses.

[0224] The cell may be a natural killer cell (or NK cell). NK cells may form part of the innate immune system. NK cells respond rapidly to endogenous signals from virus-infected cells in an MHC-dependent manner.

[0225] NK cells (belonging to the innate lymphoid cell group) are defined as large granular lymphocytes (LGL) and constitute a third cell type differentiated from a common lymphoid precursor that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, and then enter the circulation.

[0226] The cells of the invention can be any of the cell types described above.

[0227] The cells according to the invention can be generated ex vivo in hematopoietic stem cell transplants from the patient's own peripheral blood (first party), or from donor peripheral blood (second party) or peripheral blood from an unrelated donor (third party).

[0228] Alternatively, cells may be derived from ex vivo differentiation of induced or embryonic precursor cells, e.g., into T cells or NK cells. Alternatively, immortalized T cell lines may be used that retain lytic function and can act as therapeutics.

[0229] In all of these embodiments, the chimeric polypeptide-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of a number of means, including transduction with a viral vector, transfection with DNA or RNA.

[0230] The cells of the invention may be ex vivo cells from a subject. The cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The cells may be activated and / or expanded, for example by treatment with an anti-CD3 monoclonal antibody, before being transduced with a nucleic acid encoding a molecule from which the chimeric polypeptide of the first aspect of the invention is derived.

[0231] The cells of the invention can be produced by: (i) isolation of a sample containing cells from a subject or other source as listed above; and (ii) transduction or transfection of cells with one or more nucleic acid sequences encoding the chimeric polypeptide.

[0232] The cells can then be purified (eg, selected based on expression of the antigen-binding domain of the antigen-binding polypeptide).

[0233] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the invention.

[0234] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may optionally comprise one or more additional pharma- ceutically active polypeptides and / or compounds.Such a preparation may be, for example, in a form suitable for intravenous infusion.

[0235] Treatment The invention provides methods of treating disease comprising administering to a subject a cell of the invention (eg, in a pharmaceutical composition described above).

[0236] Methods of treating disease relate to therapeutic uses of the cells of the invention, where the cells can be administered to a subject already suffering from a disease or condition to alleviate, relieve or ameliorate at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0237] The method of preventing disease relates to the prophylactic use of the cells of the present invention. Herein, such cells can be administered to a subject who is not yet afflicted and / or does not show any symptoms of disease to prevent or impair the cause of disease, or to reduce or prevent the occurrence of at least one symptom associated with disease. The subject may be predisposed to disease or may be considered to be at risk of developing disease.

[0238] The method may include the following steps: (i) isolating a sample comprising cells; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering cells from (ii) to a subject.

[0239] The sample containing the cells may be isolated from a subject as described above or from another source.

[0240] The present invention provides the cells of the present invention for use in the treatment and / or prevention of disease.

[0241] The invention also relates to the use of the cells of the invention in the manufacture of a medicament for the treatment of a disease.

[0242] The disease to be treated by the method of the present invention may be a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell carcinoma), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0243] The disease can be multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), or diffuse large B-cell lymphoma (DLBCL).

[0244] The disease can be a plasma cell disorder, e.g., plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, or smoldering multiple myeloma.

[0245] The cells of the present invention may be capable of killing target cells, such as cancer cells. Target cells may be characterized by the presence of tumor-secreted ligands or chemokine ligands near the target cells. Target cells may be characterized by the presence of soluble ligands along with the expression of tumor-associated antigens (TAA) on the target cell surface.

[0246] The cells and pharmaceutical compositions of the invention may be for use in the treatment and / or prevention of the above mentioned diseases.

[0247] The present invention will now be further illustrated by examples which are intended to assist those skilled in the art in the practice of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0248] Example 1 - Expression of BCMA on the surface of multiple myeloma cells Primary myeloma cells were isolated by performing CD138 immunomagnetic selection on fresh bone marrow samples from multiple myeloma patients known to be clearly diseased. These cells were stained with the BCMA-specific J6MO mAb (GSK) conjugated with PE. Concurrently, bead standards with a known number of binding sites were generated using the PE Quantibrite bead kit (Becton Dickenson) according to the manufacturer's instructions. The BCMA copy number of myeloma cells was derived by correlating the mean fluorescence intensity from myeloma cells with a calibration curve derived from the beads. A narrow range of BCMA copy numbers on the myeloma cell surface was found: 348.7 to 4268.4 BCMA copies per cell, with a mean of 1181 and a median of 1084.9 (Figure 1). This is considerably lower than, for example, CD19 and GD2, which are classical targets of CAR.

[0249] Example 2 - Construction of an anti-BCMA FabCAR and comparison with an anti-BCMA scFv CAR A panel of nucleic acid constructs encoding CARs was generated as follows: RQR8-2A-aBCMAscFv-CD8STK-41BBZ-single chain Fv CAR construct RQR8-2A-aBCMAFAb-CD8STK-41BBZ-Fab-based CAR construct RQR8-2A-aBCMA_C11D5.3-CD8STK-41BB-z-bb2121 scFv CAR construct (positive control) The anti-BCMA scFv and Fab domains contained the VH and VL sequences of the anti-BCMA Ab1 described above (SEQ ID NOs: 5 and 6, respectively). All constructs are co-expressed with the selection-suicide gene RQR8 (described in WO2013 / 153391, with the BCMA binding portion in either scFv or Fab format).

[0250] Example 3 - CAR Expression Assay To compare the expression of different CAR formats, T cells were transduced with scFv CAR or Fab CAR and the receptors on the cell surface were detected by flow cytometry. CAR expression was detected by co-staining of T cells with soluble BCMA-FC and QBEND10 (to detect RQR8 expression).

[0251] Example 4 - FACS-based killing assay (FBK) The ability of CAR-T cells to kill target cells expressing BCMA was investigated using a FACS-based killing assay. T cells were co-cultured with target cells at effector:target ratios of 1:4 and 1:8. SupT1 cells engineered to express physiological levels of BCMA antigen were used as target cells: average 686 copies BCMA per cell (SupT1-BCMA low). Target cells expressing ultra-low levels of BCMA were also generated: average 81 copies BCMA per cell (JeKo-1). Non-engineered SupT1 cells (SupT1-NT) were also used as a negative control. 5×10 4 Assays were performed in 96-well plates in a total volume of 0.2 ml using transduced target cells / well. Co-cultures were set up after normalizing for transduction efficiency. FBK was assayed after 24 and 72 hours of incubation.

[0252] The results are shown in Figures 5 and 6. After 24 hours of co-culture (Figure 5), both BCMA scFv CAR and BCMA FabCAR successfully killed target cells expressing low levels of BCMA antigen. However, BCMA FabCAR showed better killing than the equivalent scFv CAR of target cells expressing ultra-low levels of BCMA (JeKo-1). BCMA FabCAR also showed better killing than the other BCMA scFv CAR (bb2121). After 72 hours (Figure 6), a similar pattern is observed. At an E:T ratio of 1:8, BCMA FabCAR almost completely killed ultra-low BCMA expressing target cells, whereas scFv CAR with equivalent binder showed only partial target cell killing.

[0253] Example 5 - Cytokine Release Secretion of IL-2 and interferon-γ (IFNγ) by CAR T cells was measured by collecting supernatants 24 hours after co-culture at effector:target ratios of 1:4 and 1:8 as described in Example 4. Production of IL-2 and IFN-G was detected by ELISA.

[0254] IFNγ results for co-cultures performed with antigen non-BCMA expressing target cells (FIG. 7A) or target cells expressing low levels of BCMA (FIG. 7B) are shown in FIG. 7. BCMA FabCAR expressing T cells produced similar levels of IFNγ following co-culture with BCMA low expressing target cells than the equivalent scFv CAR. The background with non-transduced SupT1 target cells for both bb2121 and the equivalent scFv CAR was higher than that seen with FabCAR.

[0255] Example 6 - Proliferation Assay (PA) To measure proliferation, the same panel of CAR-expressing T cells described in Example 4 was labeled with the dye Cell Trace Violet (CTV). T cells were incubated with CTV for 20 min at 37°C. Afterwards, cells were quenched by adding 5V of complete medium. After 5 min of incubation, T cells were washed and resuspended in 2 ml of complete medium. An additional 10 min of incubation at room temperature was performed to allow for hydrolysis of acetic acid and retention of the dye. Labeled T cells were co-cultured with non-BCMA expressing target cells (SupT1 NT), low BCMA expressing target cells (SupT1BCMAlow), ultra-low BCMA expressing target cells (JeKo-1), or MM.1s target cells at a 1:1 ratio for 96 h. 5 × 10 per well 4 Assays were performed in 96-well plates in a total volume of 0.2 ml using equal numbers of transduced T cells and target cells (ratio 1:1). After co-culture, T cells were analyzed by flow cytometry to measure the dilution of CTV that occurs upon T cell division.

[0256] Cells were also gated on CD3 and the marker RQR8, and then gated on either CD8+ or CD8- to examine whole cell counts for CD4 and CD8 T cells. The results are shown in Figure 8. BCMA FabCARs produced higher CD8 cell counts compared to equivalent scFv CARs and bb2121 after co-culture with all three BCMA-expressing target cell types (low expressing, very low expressing, and MM.1s targets), reflecting either higher proliferation and / or reduced cell death (Figure 8B). It is also noteworthy that in CD4 cells, the background proliferation of bb2121 and scFv CARs is higher than that seen with FabCARs. This may indicate an apparent higher proliferation of target cells, but actually reflects increased activation of the CAR in the absence of antigen.

[0257] Example 7 - Design and construction of antibodies with dual variable domains equivalent to the antigen-binding domain of a cleavable FabCAR A cleavable FabCAR is shown diagrammatically in FIG. 9. It contains two antigen-binding domains: an external (membrane distal) binding domain 1 and an internal (membrane proximal) binding domain 2. Binding domains 1 and 2 are linked by a cleavable linker. Cleavage of the linker, for example by matrix metalloproteinases (MMPs), removes binding domain 1 from the CAR. In the absence of cleavage (when the FabCAR is intact), the internal binding domain is partially occluded by the presence of the external domain due to steric hindrance. The internal domain is activated when the heavy and light chains are cleaved.

[0258] To demonstrate that it is possible to design a molecule with two binding domains and activate the inner binding domain by enzymatic cleavage of the outer binding domain, a soluble antibody was designed as shown in FIG. 10.

[0259] The antibody comprised a first antigen binding domain which binds BCMA and has the VH and VL sequences shown above as SEQ ID NO:29 and 30, respectively. The second antigen binding domain binds CD19 and has the VH and VL sequences shown above as SEQ ID NO:67 and SEQ ID NO:68, respectively.

[0260] An MMP-9 cleavable linker with the sequence PLGLAG (SEQ ID NO: 87) was included between the two VL domains and between the two VH domains. As a negative control, a scrambled version of this sequence was used: LALGPG (SEQ ID NO: 88).

[0261] The construct was made as follows: Plasmid 1: D8_MMP9_CAT19_HC_HuIgG1 Plasmid 2: D8_scrambled_CAT19_HC_HuIgG1 Plasmid 3: D8_MMP9_CAT19_LC Plasmid 4: D8_scrambled_CAT19_LC When CHO cells were co-transfected with either plasmid 1 and plasmid 3, they produced antibodies with MMP-cleavable linkers as shown in FIG. 10; alternatively, when co-transfected with plasmid 2 and plasmid 4, they produced equivalent antibodies with scrambled linkers in the VH- and VL-containing chains.

[0262] Example 8 - Demonstration that enzymes can be used to cleave the outer binding domain of a two binding domain antibody The antibody described in Example 7 contained an MMP-9 cleavable linker between the VH and VL domains of the BCMA binding domain and the CD19 binding domain. To demonstrate that the BCMA binding domain can be cleaved using MMP-9 enzyme, a digestion assay was set up as follows.

[0263] Recombinant human MMP-9 was prepared at 100 μg / mL in assay buffer. P-aminophenylmercuric acetate (APMA) was added to a final concentration of 1 mM to activate the enzyme and incubated at 37° C. for 24 hours.

[0264] Digestion assays were prepared by mixing 50ug antibody and 6ul activated enzyme in a final buffer volume of 200ul. Digestion assay samples were incubated at 37°C and 1ug antibody solution (4ul) was removed at various time points, resuspended in NuPAGE loading dye, boiled, frozen and then run on SDS-PAGE. Cleavage rates were analyzed by densitometry and the results are shown in Figure 11.

[0265] Antibodies containing MMP-9 cleavable linkers were digested by matrix metalloproteinase over time, reaching a plateau after approximately 1 hour of exposure to the enzyme. Antibodies with scrambled linkers did not show a significant increase in digestion, even after 24 hours of exposure to the enzyme.

[0266] Example 9 - Investigating the effect of truncation of the outer binding domain on the binding of the inner binding domain to a target antigen To investigate the activity of the internal binding domain with or without removal of the external binding domain, an ELISA assay was set up to investigate binding to CD19. Recombinant CD19 was coated on 96-well plates at 1ug / ml in 50ul / well. After blocking, antibodies described in Example 7 (containing MMP-9 linker or scrambled linker and with or without prior exposure to MMP-9) were loaded at 15ug / ml for 30 minutes and then washed with PBS 0.05% Tween 20. Secondary anti-human Fc HRP-conjugated antibody was added at 1:3000 dilution and incubated for 1 hour. Signal was detected with 1-step Ultra TMB substrate and blocked in 1M H2SO4. The results are shown in Figure 12: the soluble antibody (D8-MMP-CAT19) showed increased binding to CD19 upon MMP-9 cleavage, whereas for the antibody with a scrambled linker (D8-scrambled-CAT19), CD19 binding was unaffected by prior exposure to MMP-9.

[0267] Example 10 - Investigating the effect of exo-binding domain truncation on the kinetics of CD19 binding CD19 binding kinetics was tested by capturing antibodies on a Protein A chip with a Biacore8K instrument. Antibodies described in Example 8 (with or without pre-exposure to MMP-9) and a positive control antibody (CAT19 IgG) were captured at 75 RU for IgG and 100 RU for the two binding domain constructs. Recombinant CD19 was dialyzed into HBS-EP+ buffer and analytes were tested at 500 nM using 2-fold serial dilutions. Results are shown in Figure 13. Antibodies with MMP-9 cleavable linkers had increased Rmax of CD19 binding after cleavage with MMP-9 (D8-MMP9-CAT19 cleavage) compared to equivalent antibodies that were not exposed to MMP-9 (D8-MMP9-CAT19).

[0268] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of molecular biology or related fields are intended to be within the scope of the following claims.

Claims

[Claim 1] The invention as depicted in the drawings.

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