Antibodies and car-ts against HLA-DP for treatments

Abstract

Chimeric antigen receptors (CARs) that bind to Human Leukocyte Antigen (HLA)-pan DP, cells that comprise the CARS and methods of making and using the cells are provided.

Description

ANTIBODIES AND CAR-TS AGAINST HLA-DP FOR TREATMENTSCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 711,071, filed October 23, 2024, which is incorporated by reference for all purposes.BACKGROUND OF THE INVENTION

[0002] Relapse of hematological malignancies often originates from residual cancer and / or its initiating cells. Target antigens on cells, such as CD19 and BCMA, are often downregulated after immunotherapy (Orlando et al., Nature Medicine 2018; Dhodapkar et al., Blood Cancer Discovery 2022).BRIEF SUMMARY OF THE INVENTION

[0003] In some embodiments, a human cell comprising a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises an extracellular binding domain that specifically binds to Human Leukocyte Antigen (HLA)-DP, wherein the extracellular binding domain is linked to a transmembrane domain and optionally at least one intracellular signaling domain. In some embodiments, the human cell is a T-cell, natural killer (NK) cell, lymphocyte or a macrophage. In some embodiments, the extracellular binding domain comprises a heavy chain variable region and a light chain variable region from an antibody that binds to HLA-DP.

[0004] In some embodiments, (a) the heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HDR2, wherein HCDR1 comprises SEQ ID NO: 2, HCDR2 comprises SEQ ID NO:3 and HCDR3 comprises SEQ ID NO:4; and / or (b) the light chain variable region comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LDR2, wherein LCDR1 comprises SEQ ID NO: 6, LCDR2 comprises SEQ ID NO:7 and LCDR3 comprises SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90, 95, 98, 99 or 100% identicalto SEQ ID NO:1 and / or the light chain variable region comprises an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO:5.

[0005] In some embodiments, the extracellular binding domain is a scfv.

[0006] In some embodiments, the intracellular signaling domain is an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the CAR comprises two or more intracellular signaling domains. In some embodiments, the intracellular signaling domain(s) is(are) selected from the group consisting of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcRbeta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10 and DAP12.

[0007] In some embodiments, at least one intercellular signaling domain is a co-stimulatory domain. In some embodiments, the co-stimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0008] Also provided are nucleic acids comprising a coding sequence encoding the CAR as provided above or elsewhere herein.

[0009] Also provided are expression cassettes comprising a promoter operably linked to the coding sequence as described above or elsewhere herein.

[0010] Also provided is a vector comprising the nucleic acid or the expression cassette as described above or elsewhere herein. In some embodiments, the vector is a viral vector.

[0011] Also provided is a method of making a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the method comprises, introducing the nucleic acid or the expression cassette, or the vector as provided above or elsewhere herein into the cell such that the cell expresses the CAR. In some embodiments, the introducing is in vitro or ex vivo. In some embodiments, the method further comprises, before the introducing, isolating the cell from a human. In some embodiments, the cell is a T-cell, natural killer (NK) cell, lymphocyte or a macrophage.

[0012] Also provided is a method of killing a cancer cell expressing HLA-DP in a human. In some embodiments, the method comprises administering the human cell as described above or elsewhere herein to the human, wherein at least one cancer in the human is killed. In some embodiments, further comprising before the administering, isolating a cell from the human; and expressing the CAR in the cell. In some embodiments, the human has received radiation therapy to kill hematopoietic stem cells prior to the administering. In some embodiments, the hematopoietic stem cells are administered to the human before, with or after the administering of the human cell. In some embodiments, the hematopoietic stem cells administered to the human are autologous or allogeneic to the human. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is a B-cell lymphoma.

[0013] Also provided is a bispecific antibody that specifically binds to Human Leukocyte Antigen (HLA)-DP and a second target. In some embodiments, the second target is CD3. In some embodiments, the bispecific antibody comprises (i) a first polypeptide comprising an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO: 22 and (ii) a second polypeptide comprising an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO: 23.

[0014] Also provided is a nucleic acid comprising a coding sequence encoding the first polypeptide or the second polypeptide making up the bispecific antibody, or both the first polypeptide and the second polypeptides, as described above or elsewhere herein.

[0015] Also provided is an expression cassette comprising a promoter operably linked to the coding sequence as described above or elsewhere herein.

[0016] Also provided is a vector comprising the nucleic acid or the expression cassette as described above or elsewhere herein. In some embodiments, the vector is a viral vector.

[0017] Also provided is a method of making a cell expressing a bispecific antibody that specifically binds to Human Leukocyte Antigen (HLA)-DP and CD3. In some embodiments, the method comprises introducing the nucleic acid as described above or elsewhere herein, or the expression cassette as described above or elsewhere herein, or the vector as described above or elsewhere herein into the cell such that the cell expresses the bispecific antibody.

[0018] Also provided is a method of killing a cancer cell expressing HLA-DP in a human. In some embodiments, the method comprises administering the bispecific antibody as described above or elsewhere herein to the human, wherein at least one cancer in the human is killed. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is a B-cell lymphoma.DEFINITIONS

[0019] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0020] As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid-derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0021] The term “bispecific antibody” as used herein, refers to an antibody that binds to two or more different epitopes. In some embodiments, a bispecific antibody binds to epitopes for two different target antigens. In some embodiments, a bispecific antibody binds to two differentepitopes for the same target antigen. Bi-specific antibodies can be made in a number of ways. See, e.g., Brinkmann U, Kontermann RE. The making of bispecific antibodies. mAbs 2017; 9: 182-212 and FIG. 10A-B. In some embodiments, the bi-specific antibodies described herein are diabodies or knob-in-a-hole IgG antibodies or otherwise use knob-in-a-hole technology. See, e.g., Xu, et al., MAbs 7(1):231-42 (2015).

[0022] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific or multivalent antibodies formed from antibody fragments. A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. A F(ab')2 fragment has a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known. An "Fv" is a minimal antibody fragment that contains a complete antigen-recognition and binding site and is a dimer of one heavy- and one light-chain variable region domain.

[0023] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgGa, IgGr. The antibodies described herein can be of any of these classes or subclasses.

[0024] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4.

[0025] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions that interrupt the four "framework" regions of s variable domain. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each heavy or light chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

[0026] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., North, Kabat, Chothia, internationalImMunoGeneTics database (IMGT), and AbM (see, e.g., North method, (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011; Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)). Definitions of CDRs are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al., Methods Enzymol., 203, 121-153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs herein refer to CDRs determined according to the method of North (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011) unless indicated otherwise. The CDRs described herein were determined using the IGBLAST algorithm with standard settings. See, e.g., Ye et al., Nucleic Acids Res. 2013 Jul;41(Web Server issue): W34-40.

[0027] “Epitope" or "antigenic determinant" as used in the present disclosure in the context of antibody binding refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids and / or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Binding of an antibody to an epitope can be influenced by other environmental factors, such as the presence of calcium ions.

[0028] As used herein, the term “specifically binds” to a target, e.g., human anti-HLA-pan DP, refers to a binding reaction whereby the antibody binds to the target with greater affinity, greateravidity, and / or greater duration than it binds to a different target. In some embodiments, a targetbinding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. The term “specific binding,” “specifically binds to," or “is specific for" a particular target, as used herein, can be exhibited, for example, by a molecule (e.g., an antibody) having an equilibrium dissociation constant KD for the target of, e.g., 10'2M or smaller, e.g., 10'3M, 10'4M, IO’5M, W6M, IO’7M, IO'8M, 10’9M, IO’10M, IO’11M, or IO’12M. In some embodiments, an antibody has a KD of less than 100 nM or less than 10 nM.

[0029] The term “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0030] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0031] The term "effective amount," as used herein, refers to that amount of cell that expressed a CAR comprising an anti-HLA-pan DP binding protein, that is sufficient to effect treatment of adisease associated with the growth and / or proliferation of cancer cells, as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The dosages for administration can range from, for example, 5×105CAR-positive T-cells per kg to 2×107CAR-positive T-cells per kg of a CAR-expressing cell as described herein. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding portion thereof are minimized and / or outweighed by the beneficial effects.

[0032] The terms "identical" or “percent identity,” in the context two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (i.e., about 60% identity, preferably at least 65%, 70%, 75%, 80%, or 85% identity; and often at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithm (e.g., a BLASTP algorithm with default parameters for comparison of two polypeptide sequences. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. The algorithms can account for gaps and the like. Percent identity is over the entire length of the reference sequence unless indicated otherwise.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 A-D. Cytotoxicity of anti-HLA-DP CAR-T against various hematological malignant cell lines. (A) JeKo-1 of wild and HLA-DP KO types; (B) JJN3 of wild and HLA-DP KO types; (C) Namalwa; (D) Toledo.

[0034] FIG. 2A-C. In vivo cytotoxicity of anti-HLA-DP CAR-T against JeKo-1. (A) Murine study design and bioluminescent imaging. Disseminated JeKo-1 lymphoma model implanted intravenously in NSG mice, n = 6 / arm. Anti-HLA-DP CAR-T was evaluated (Group 1) compared to Empty (no binder sequence) CAR-T (Group 2), anti-CD19 CAR-T (Group3,Kymriah) and no treatments (Group 4); (B) Time course of BLI in each mouse group; (C) Survival comparison between each group.FIG. 3. Bispecific construct is depicted.FIG. 4. Positive staining by flow cytometry.FIG. 5. Cytotoxicity of bispecific antibodies (BsAbs) against JJN-3DETAILED DESCRIPTION OF THE INVENTION

[0035] The inventors have discovered that cells expressing a chimeric antigen receptor (CAR) that target Human Leukocyte Antigen (HLA)-DP can be used to effectively kill cancerous B-cells, including for example lymphoma cells including diffuse large B-cell lymphoma (DLBCL) cells and follicular lymphoma cells as well as leukemia cells. While particular antibody variable regions were used in the examples to demonstrate targeting and killing of cancer cells expressing HLA-DP, it is expected other ligands and other antibody sequences that specifically target HLA-DP can also be used as an extracellular HLA DP binding domain of CAR-expressing cells as described herein. Also provided are bispecific antibodies that target Human Leukocyte Antigen (HLA)-DP and a second target. These can be used for example as BiTE (bispecific T cell engager) molecules.

[0036] HLA DP is a heterodimer of a and P chains encoded by the HLA DP locus and that is expressed in all stages of B-cell maturation. See, e.g., Alvarez et al., Mol. Cell. Proteomics 18, 2459-2477 (2019); Refsgaard et al., ImmunoInformatics. 1-2, 100005 (2021); Van Balen et al., J. Immunol. 204, 3273-3282 (2020); and Nilsson et al., SciAdv. 2023 Nov 24; 9(47). The HLA-DP A gene (DPA1) encodes the a chain of the mature class II molecule, and has limited sequence diversity. The HLA DP B gene (DPB1) has a highly polymorphic exon 2 that gives rise to the P chain of the molecule. The a chain is noncovalently bound to the P chain. An exemplary HLA DP alpha chain protein encoded by the DPA1 *01:03:01:01 allele is provided as SEQ ID NO: 9. Other HLA DP alpha chain alleles, and the proteins encoded by the alleles, are known and include but are not limited to, for example, DPAl*01:03:02, DPAl*01:04:01, DPAl*01:05, DPAl*01:06:01, DPAl*01:07, DPAl*01:08, DPAl*01:09, DPAl*01:10, DPAl*01:ll:01, DPAl*01:12:01, DPAl*01:13, DPA1*O1:14, DPAl*01:15, DPA1*O1:16, DPAl*01:17, DPA1*O1:18, DPA1*O1:19, DPAl*01:20, DPA1*O1:21Q, DPA1*O1:22, DPAl*01:23,DPA1 *01:24, DPA1 *01:25, DPA1 *01:26, DPA1 *01:27, DPA1 *01:28, DPA1 *01:29N, DPAl*01:30, DPAl*01:31, DPA1*O1:32N, DPAl*01:33:01, DPAl*01:34, DPA1*O1:35N, DPA1*O1:36, DPA1*O1:37, DPA1*O1:38, DPA1*O1:39, DPAl*01:40, DPAl*01:41:01, DPAl*01:42:01:01, DPAl*01:42:01:02, DPA1 *01:43, DPA1*O1:44, DPA1*O1:45, DPA1*O1:46, DPA1*O1:47, DPA1*O1:48, DPA1*O1:49, DPAl*01:50, DPAl*01:51, DPA1*O1:52, DPAl*01:53, DPA1*O1:54, DPAl*01:55N, DPA1*O1:56, DPAl*01:57, DPAl*01:58:01:01, DPAl*01:58:01:02, DPA1*O1:59, DPAl*01:60, DPA1*O1:61, DPA1*O1:62, DPAl*01:63, DPA1*O1:64, DPA1*O1:65, DPA1*O1:66N, DPA1*O1:67, DPA1*O1:68, DPA1*O1:69, DPAl*01:70, DPA1*O1:71, DPA1*O1:72, DPAl*01:73, DPA1*O1:74, DPA1*O1:75, DPA1*O1:76, DPAl*01:77:01:01, DPA1 *01:77:01:02, DPA1*O1:78, DPA1*O1:79N, DPA1*O1:8O, DPA1*O1:81, DPA1*O1:82, DPAl*01:83, DPA1*O1:84, DPA1*O1:85, DPA1*O1:86, DPA1*O1:87Q, DPA1*O1:88, DPA1*O1:89, DPAl*01:90, DPA1*O1:91, DPA1*O1:92, DPA1*O1:93, DPA1*O1:94, DPAl*01:95, DPA1*O1:96, DPA1*O1:97, DPA1*O1:98, DPA1*O1:99, DPAl*01:100, DPAl*01:101, DPAl*01:102, DPAl*01:103, DPAl*01:104, DPAl*01:105, DPAl*01:106, DPAl*01:107:01:01, DPAl*01: 107:01:02, DPAl*01:108, DPAl*01:109, DPAl*01:110, DPA1 *01:111, DPA1 *01:112, DPAl*01:113, DPAl*01:l 14N, DPAl*01:115, DPA1*O1:116, DPA1*O1:117, DPAl*01:118, DPA1*O1:119N, DPAl*01:120N, DPA1*O1:121, DPA1*O1:122, DPA1*O1:123N, DPA1*O1:124, DPA1*O1:125, DPA1*O1:126, DPA1*O1:127, DPAl*01:128:01:01, DPAl*01:128:01:02, DPA1*O1:129, DPAl*01:130, DPAl*01:131, DPA1*O1:132, DPAl*01:133, DPA1*O1:134, DPAl*01:135, DPAl*01:136, DPA1*O1:137N, DPAl*01:138, DPAl*01:139, DPAl*01:140, DPA1*O1:141, DPA1*O1:142, DPA1*O1:143, DPA1*O1:144, DPA1*O1:145, DPA1*O1:146, DPA1*O1:147N, DPA1*O1:148, DPA1*O1:149, DPAl*01:150, DPAl*01:151, DPA1*O1:152, DPAl*01:153, DPA1*O1:154N, DPAl*01:155:01:01, DPAl*01:155:01:02, DPA1*O1:156, DPA1*O1:157, DPAl*01:158, DPA1*O1:159, DPAl*01:160, DPA1*O1:161, DPA1*O1:162, DPAl*01:163Q, DPA1*O1:164, DPA1*O1:165, DPA1*O1:166, DPA1*O1:167, DPA1*O1:168N, DPA1*O1:169, DPAl*01:170, DPA1*O1:171Q, DPA1*O1:172, DPA1*O1:173, DPA1*O1:174, DPAl*01:175, DPA1*O1:176, DPA1*O1:177, DPAl*01:178, DPA1*O1:179, DPAl*01:180, DPAl*01:181, DPA1*O1:182, DPAl*01:183, DPA1*O1:184, DPA1*O1:185, DPA1*O1:186, DPAl*01:187, DPA1*O1:188, DPA1*O1:189, DPAl*01:190, DPA1*O1:191, DPA1*O1:192, DPAl*01:193, DPA1*O1:194,DPA1*O1:195, DPA1*O1:196, DPA1*O1:197, DPA1*01:198, DPA1*O1:199, DPA1*01:200, DPA1*01:201, DPA1*01:202, DPA1*01:203, DPA1*01:204, DPA1*01:205, DPA1*01:206, DPA1*01:207, DPA1*01:208, DPA1*02:01:01:01, DPA1*02:01:03, DPA1 *02:01:04, DPA1 *02:01:05, DPA1 *02:01:06, DPA1 *02:01:07, DPA1*02:01:08:01, DPA1*02:01:09, DPAl*02:01:10, DPA1 *02:01: 11, DPA1 *02:01:12, DPA1*02:01:13, DPA1 *02:01: 14, DPA1 *02:01: 15, DPA1 *02:01: 16, DPA1*02:01:17, DPA1 *02:01: 18, DPA1*02:01:19, DPA1 *02:01:20, DPA1*02:01:21, DPA1 *02:01:22, DPA1 *02:01:23, DPA1 *02:01:24, DPA1*02:01:25, DPA1 *02:01:26, DPA1 *02:01:27, DPA1*02:01:28, DPA1 *02:01:29, DPA1*02:01:30, DPA1*02:01:31, DPA1*02:02:02:01, DPA1 *02:02:03, DPA1 *02:02:04, DPA1*02:02:05, DPA1 *02:02:06, DPA1 *02:02:07, DPA1*02:02:08, DPA1 *02:02:09, DPA1*02:02:10, DPA1 *02:02: 11, DPA1 *02:02: 12, DPA1*02:02:13, DPA1*02:02:14, DPA1*02:02:15, DPA1*O2: O3: O1, DPA1 *02:03:02, DPA1*02:03:03, DPA1 *02:03:04, DPA1*02:03:05, DPA1*02:04, DPA1*02:05, DPA1*02:06, DPA1*02:07:01:01,DPA1 *02:07:01:02, DPA1*02:07:01:03, DPA1 *02:07:01:04, DPA1 *02:07:02, DPA1*02:07:03, DPA1 *02:07:04, DPA1*02:08, DPA1*02:09:01:01, DPA1 *02:09:01:02, DPA1*02:09:01:03, DPA1*02:09:01:04, DPA1 *02:09:02, DPA1*02:10, DPA1*O2:11, DPA1*O2: 12:01, DPA1 *02: 12:02, DPA1*02:13N, DPA1*O2:14, DPA1*02:15, DPA1*02:16, DPA1*02:17, DPA1*O2:18, DPA1*O2:19, DPA1*02:20, DPA1*02:21:01, DPA1 *02:21:02, DPA1 *02:21:03, DPA1*02:21:04, DPA1 *02:22:01, DPA1 *02:22:02, DPA1*02:23:01, DPA1 *02:23:02, DPA1*O2:24, DPA1*O2:25, DPA1 *02:26:01:01, DPA1 *02:26:01:02, DPA1 *02:27:01, DPA1*02:27:02, DPA1*02:27:03, DPA1*02:28:01, DPA1*02:28:02, DPA1*O2:29, DPA1*02:30, DPA1*O2:31, DPA1*O2:32N, DPA1*O2:33, DPA1*02:34:01:01,DPA1 *02:34:01:02, DPA1*02:35, DPA1*O2:36, DPA1*O2:37, DPA1*02:38Q, DPA1*O2:39, DPA1*02:40, DPA1*O2:41N, DPA1*O2:42, DPA1*O2:43, DPA1*O2:44, DPA1*02:45:01, DPA1 *02:45:02, DPA1*02:45:03, DPA1*O2:46, DPA1*02:47:01, DPA1 *02:47:02, DPA1*O2:48, DPA1*O2:49, DPA1*02:50:01, DPA1*02:50:02, DPA1*O2:51, DPA1*O2:52N, DPA1*O2:53, DPA1*O2:54, DPA1*O2:55, DPA1*O2:56Q, DPA1*O2:57, DPA1*02:58, DPA1*02:59:01, DPA1*02:59:02, DPA1*02:60, DPA1*O2:61, DPA1*02:62, DPA1*O2:63, DPA1*O2:64Q, DPA1*O2:65, DPA1*02:66:01N, DPA1*02:66:02N, DPA1*O2:67, DPA1*O2:68, DPA1*02:69:01, DPA1 *02:69:02, DPA1*02:70, DPA1*O2:71, DPA1*O2:72, DPA1 *02:73, DPA1*O2:74N, DPA1 *02:75, DPA1 *02:76, DPA1 *02:77, DPA1 *02:78,DPA1 *02:79, DPA1 *02:80N, DPA1 *02:81, DPA1 *02:82, DPA1 *02:83, DPA1 *02:84, DPA1*O2:85, DPA1*O2:86, DPA1*O2:87, DPA1*O2:88, DPA1*O2:89, DPA1*02:90, DPA1*O2:91, DPA1*O2:92, DPA1*O2:93, DPA1*O2:94N, DPA1*O2:95, DPA1*O2:96, DPA1*O2:97, DPA1*O2:98, DPA1*O2:99, DPA1*02:100, DPA1*02:101:01, DPA1*02:101:02, DPA1*02:102Q, DPA1*02:103, DPA1*02:104, DPA1*02:105, DPA1*02:106, DPA1*02:107, DPA1*02:108N, DPA1*02:109, DPAl*02:110:01, DPAl*02:110:02, DPA1*O2:111, DPA1 *02: 112, DPA1 *02: 113, DPA1 *02: 114, DPA1 *02: 115, DPA1 *02: 116, DPA1 *02: 117, DPA1*O2:118, DPA1*O2:119, DPA1*02:120, DPA1*O2:121, DPA1*O2:122, DPA1*O2:123, DPA1*O2:124, DPA1*O2:125, DPA1*O2:126, DPA1*O2:127, DPA1*O2:128, DPA1*O2:129, DPA1*02:130, DPA1*02:131, DPA1*O2:132, DPA1*O2:133N, DPA1*O2:134, DPA1*O2:135, DPA1*O2:136, DPA1*02:137, DPA1*O2:138, DPA1*03:01:01:01, DPA1 *03:01:01:02, DPAl*03:01:01:03, DPA1 *03:01:01:04, DPA1 *03:01:01:05, DPA1 *03:01:01:06, DPAl*03:01:01:07, DPAl*03:01:01:08, DPAl*03:01:01:09, DPAl*03:01:01:10, DPAl*03:01:01:ll, DPAl*03:01:01:12, DPAl*03:01:01:13, DPAl*03:01:01:14, DPAl*03:01:01:15, DPAl*03:01:02:01, DPA1 *03:01:02:02, DPA1 *03:01:02:03, DPA1*03:01:02:04, DPA1 *03:01:03, DPA1*03:01:04, DPA1*03:02, DPA1*03:03, DPA1*03:04, DPA1*03:05:01:01Q, DPA1*03:05:01:02Q, DPA1*O3: O5: O2Q, DPA1*03:06:01, DPA1*03:06:02, DPA1 *03:06:03, DPA1*03:07:01, DPA1*03:07:02, DPA1*03:08, DPA1 *03:09, DPA1*03:10N, DPA1*O3:11N, DPA1*O3:12, DPA1*03:13, DPA1*O3:14, DPA1*03:15, DPA1*O3:16, DPA1*O3:17, DPA1*03:18, DPA1*O3:19N, DPA1*04:01:01:01, DPA1*04:01:01:02, DPA1*04:01:01:03, DPA1 *04:01:02, DPA1*04:02:01:01, DPA1*04:02:01:02, DPA1*O4: O3, DPA1*04:04, DPA1*04:05, DPA1*04:06, or DPA1*04:07 or variants thereof have been described and are known in the art.

[0037] The present disclosure provides chimeric antigen receptors (CARs) that comprising an extracellular HLA-DP binding protein wherein the CAR is expressed in cells. Also provided are for example nucleic acids encoding the CARs. Chimeric antigen receptors (CARs) are recombinant receptor constructs comprising an extracellular antigen-binding domain (e.g., a HLA-DP -binding domain from an antibody as described herein) joined to a transmembrane domain, and further linked to an intracellular signaling domain (e.g., an intracellular T cell signaling domain of a T cell receptor) that transduces a signal to elicit a function. In certain embodiments, immune cells (e.g., T cells or natural killer (NK.) cells or macrophages) aregenetically modified to express CARs that comprise one or more HLA-DP -binding domains of the antibodies described herein and optionally have the functionality of effector cells (e.g., T cell cytotoxic functions).

[0038] In some standard CAR embodiments, the components include an extracellular targeting domain comprising an HLA DP -binding variable region as described herein, a transmembrane domain and intracellular signaling / activation domain, which are typically linearly constructed as a single fusion protein. The "transmembrane domain" is the portion of the CAR that links the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the host cell that is modified to express the CAR, e.g., the plasma membrane of an immune effector cell. The intracellular region may contain a signaling domain of TCR complex, and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a "first-generation CAR" generally has a CD3-zeta signaling domain. Additional costimulatory intracellular domains may also be introduced (e.g., second and third generation CARs) and further domains including homing and suicide domains may be included in CAR constructs. CAR components are further described below.

[0039] A CAR construct encoding a CAR may also comprise a sequence that encodes a signal peptide to target the extracellular domain to the cell surface.

[0040] The extracellular domain comprises one or more anti-HLA DP -binding polypeptide (e.g., an anti-HLA DP-binding domain). Any polypeptide that specifically binds to HLA DP in the target individual can be used. In some embodiments, the HLA DP binding polypeptide comprises a single chain antibody (scFv) or a heavy chain variable region (e.g., a VHH, e.g., such as a nanobody™). ScFvs can be constructed as described in the art, e.g., such that the heavy chain variable region and the light chain variable region are covalently-linked in either orientation directly or via a peptide linker. In some embodiments, the HLA DP binding polypeptide binds to at least the HLA DP in the individual who will receive the cell expressing the CAR. Because the HLA DP alpha chain does not have significant variation, in some embodiments, the HLA DP binding polypeptide is an HLA pan DP binding protein, i.e., that binds to a common epitope among two or more different HLA DP alpha chain protein variants. An HLA pan DP binding protein is a protein that binds to two or more different HLA DP alphachain variants. Because the alpha chain does not greatly vary there are a number of conserved regions within the HLA DP alpha chain protein to which an HLA pan DP binding polypeptide can be targeted. In some embodiments, the HLA pan DP alpha chain binding polypeptide binds to SEQ ID NO:7 and / or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more alpha chains for example as encoded by the DPA1 alleles recited in the paragraph above.

[0041] In some embodiments, the HLA pan DP alpha chain binding polypeptide comprises an scFv comprising a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1, HCDR2 and HDR2, wherein HCDR1 comprises SEQ ID NO: 2, HCDR2 comprises SEQ ID NO:3 and HCDR3 comprises SEQ ID NO:4; linked to a light chain variable region comprising a light chain complementarity determining region (LCDR) 1, LCDR2 and LDR2, wherein LCDR1 comprises SEQ ID NO: 6, LCDR2 comprises SEQ ID NO:7 and LCDR3 comprises SEQ ID NO:8, wherein the CDR sequences are determined by IGBLAST. An exemplary scFv sequence comprising the above CDRs is SEQ ID NO: 10. While SEQ ID NO: 11 is used as a linker between VH and VI regions, it will be appreciated that other amino acid linkers (e.g., between 1-50 amino acids in length) linkers can also be used. In some embodiments, CDRs from any antibody that binds to HLA DP can be introduced into a humanized framework region to form a scFv as an extracellular region of a CAR as described herein. Exemplary antibodies that bind to HLA DP include for example, Rabbit monoclonal [SP228] to HLA-DP (commercially available from Abeam), HLA-DP Recombinant Rabbit Monoclonal Antibody (PD00-70) (Thermo Fischer Scientific), HLA-DP (MHC II) Recombinant Rabbit Monoclonal Antibody (HLA-DPB1 / 2862R) (Thermo Fischer Scientific), HLA-DP (MHC II) Monoclonal Antibody (BRA-FB6) (Thermo Fischer Scientific), HLA-DP (MHC II) Monoclonal Antibody (SPM421) (Thermo Fischer Scientific). Humanized light and heavy chain variable regions from these antibodies can be formed into an scFv and form part of the extracellular domain of a CAR.

[0042] In some embodiments, any one or more of the above-listed CDR sequences include one or two amino acid changes, which in some embodiments are conservative amino acid changes. The variable regions will include the CDRs as well as framework sequences adjacent to the CDRs. In some embodiments, the framework sequences can be selected to avoid human immune response, e.g., the variable regions can be “humanized.” Exemplary the heavy chain variableregion and light chain variable regions comprising the above-listed CDR sequences are SEQ ID NO:1 and SEQ ID NO:5, respectively. In some embodiments, the humanization is uses one or more of the following framework sequences: IGHV3-6601, IGHJ401, IGKV1-2701, and IGKJ401 with the CDRs inserted into these frameworks. Humanization methods are described in, but not limited to, Yi-Fan Zhang & Mitchell Ho (2017) mAbs, 9:3, 419-429.

[0043] In some embodiments, the CAR may contain one or more hinge domains that link the antigen binding domain comprising the anti-HLA-pan DP -binding domain and the transmembrane domain for positioning the antigen binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunoglobulin hinge region, or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1, CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered.

[0044] Any transmembrane suitable for use in a CAR construct may be employed. Such transmembrane domains, include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, eg., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD Ila, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

[0045] A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.

[0046] A CAR construct of the present disclosure can include one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte). The intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. In one embodiment, a costimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co-stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e g., T cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0047] Examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0048] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosinebased activation motifs or ITAMs.

[0049] Examples of ITAM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta.

[0050] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domainof the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4- IBB (CD 137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0051] In some embodiments, a CAR of the disclosure comprises a signal sequence, an scFv that binds HLA DP (e.g., SEQ ID NO: 10), optionally an IgG4 (EQ) Spacer, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z domain. In some embodiments, the CAR comprises SEQ ID NO: 12, optionally lacking the 4XGS Linker and / or GFP portions in SEQ ID NO: 12.

[0052] In some embodiments, a CAR may be designed as an inducible CAR, or may otherwise comprise a mechanism for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment. Thus, for example, in some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657.

[0053] In some embodiments, a cell expressing a CAR comprising one or more HLA DP-binding domains as described herein also expresses a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target.

[0054] In some embodiments, a host cell, e.g., a host T cell is modified to express a HLA DP-binding domain as described herein, or a chimeric molecules, such as a chimeric receptor comprising such a domain, using a gene editing system, such as a Cas / CRISPR system, a Transcription activator-like effector nuclease (TALEN) system, a homing endonuclease (HE) system, or a zinc-finger nuclease (ZFN) system.

[0055] The mammalian cell expressing the CARs described herein are not limited by the type of cells (which can be immune cells) genetically modified to express a CAR. Illustrative immune cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes. In some embodiments, the T cells are CD8+ T cells Treg cells. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient to undergo immunotherapy. In some embodiments, the immune cells are allogeneic. Methods of making CAR-expressing cells are described, e.g., in US2016 / 0185861 and US2019 / 0000880.

[0056] Many methods for introducing nucleic acids and viral vectors (e.g., viral particles) into a target cell (e.g., a CD8+T cell) are available. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, a viral vector may be used, such as an adenovirus, adeno-associated virus (AAV), lentivirus vector, a vaccinia virus vector, or any of a number of different vectors.

[0057] In certain embodiments, the CARs comprising the HLA DP -binding domain described herein or variants thereof, are recombinantly expressed using methods well known to those of skill in the art. For example, using the sequence information provided herein, nucleic acids encoding the desired CAR can be prepared according to a number of standard methods known tothose of skill in the art. The nucleic acids are transfected into host cells that then express the desired CAR.

[0058] Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, U. S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033. In addition, detailed protocols for the expression of antibodies are also provided by Liu et al. (2004) Cancer Res. 64: 704-710, Poul et al. (2000) J. Mol. Biol. 301: 1149-1161, and the like.

[0059] In some embodiments, instead of a CAR, the HLA DP binding polypeptide is part of a bi-specific or multi-valent antibody. As an example, in some embodiments, the bi-specific antibody comprises heavy and light chain variable regions that bind to HLA DP and heavy and light chain variable regions that bind to CD3 or another target protein. In some embodiments, the bi-specific antibody is a bi-specific T-cell engager (biTE) fusion protein having two scFvs, e.g., an HLA DP-specific binding fragment that targets HLA DP that is combined with a binding domain that binds to and modulates activity of a molecule on a leukocyte, such as a T-cell receptor or NK cell receptor. Such molecules include, for example, CD3 and Fc receptor molecules, among others.

[0060] An antibody construct may be “bispecific,” i.e., the antibody orantibody protein product binds two different targets (e.g., CD3 and a second, different target). A “bispecific” antibody or antibody-like product generally comprises a first binding domain and a second binding domain, wherein the first binding domain binds to HLA DP, and the second binding domain binds to another antigen or target (e.g., CD3). Multispecific antibody constructs,such as trispecific antibody constructs (including three binding domains) or constructs having more than three (e.g. four, five, or more) specificities can also be made. Exemplary bi-specific antibodies can include those having CD3 binding domains such as described in WO-2022046651 or as otherwise known, and including a binding domain that binds to HLA DP as described herein. Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015) and International Patent Publication No. WO 2015149077, describe various bispecific formats that can be used with the HLA DP biding domains described herein. Examples of bispecific antibody constructs include, but are not limited to, diabodies, single chain diabodies, tandem scFvs, bispecific T cell engager (BiTE®) format (a fusion protein consisting of two single-chain variable fragments (scFvs) joined by a linker), BsAb fragments (e.g., bispecific single chain antibodies), bispecific fusion proteins (e.g., antigen binding domains fused to an effector moiety), and Fab2 bispecifics (collectively also termed “bispecific antibody protein products”). See, e g., Chames & Baty, 2009, mAbs 1 [6]: 1-9; and Holliger & Hudson, 2005, Nature Biotechnology 23[9]i 1126-1136; Wu et al., 2007, Nature Biotechnology 25

[0011] : 1290-1297; Michaelson et al., 2009, mAbs 1 [2]: 128-141; International Patent Publication No. WO 2009032782 and WO 2006020258; Zuo et al., 2000, Protein Engineering 13 [5]:361 -367; U. S. Patent Application Publication No. 20020103345; Shen et al., 2006, J Biol Chem 281

[0016] : 10706-10714; Lu et al., 2005, J Biol Chem 280[20j: 19665-19672; and Kontermann, 2012 MAbs 4(2): 182.

[0061] The cells expressing a CAR. as described herein, or a bi-specific antibody as described herein, can be used to treat cancer, i.e., cancer cells that express HLA DP. Exemplary cancer cells that express HLA DP can include any cancer cell in a B-cell lineage, for example but not limited to leukemia or lymphoma. In some embodiments, the cancer cells are follicular lymphoma or diffuse large B-cell lymphoma cells. In some embodiments, the human receiving the cells expressing the CAR have received a treatment (e.g., within 2, 4, 6, or 8 weeks earlier) such that bone marrow stem cells have been killed, e.g., by radiation and / or chemotherapy. In some embodiments, following administration of the cells expressing the CAR (e.g., after 2, 4, 6, or 8 or more weeks after CAR cell administration), the individual receive a hematopoietic stem cell transplantation. Sources of hematopoietic stem cells for transplantation can include for example bone marrow, recruited peripheral blood and umbilical cord blood and can be autologous or allogeneic (optionally HLA-matched between the donor and recipient).EXAMPLESExample 1

[0062] Methods: We obtained a hybridoma cell line (clone B7 / 21) producing anti-HLA-pan-DP monoclonal antibody (mAb) and identified the CDR sequences of the mAb's VH and VL chains. We inserted these into an IgG4 (EQ) backbone plasmid to create an expression vector (Mandal et al., Nature Cancer 2023). The resulting CAR comprises an scFv of the heavy and light chain domains of (SEQ ID NO: 1 and SEQ ID NO:5) linked by a peptide linker, linked to an IgG4 EQ hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain and a CD3-zeta domain. The CAR’s amino acid sequence was SEQ ID NO: 12. Lentiviral transduction of primary human T cells from a healthy donor created CAR-T cells. In vitro cytotoxic assays were preformed with various lymphoma, multiple myeloma (MM), and blastic plasmacytoid dendritic cell neoplasm (BPDCN) cell lines, as well as primary lymphoma cells. An in vivo mouse experiment was performed using NSG mice.

[0063] Results: Analyzing CoMMpass mRNA data (release IA19), we evaluated HLA-DP expression of myeloma cells derived from patients. Surprisingly, HLA-DP was significantly higher at relapse compared to diagnosis (P < 0.0001), as well as in t(4;14) positive cases (P=0.007), but not in del-17p and Iq-gain. HLA-DP CARs were efficacious in in vitro cytotoxicity assays targeting lymphoma cell lines such as JeKo-1, Namalwa, and Toledo, the MM cell line JJN-3 (FIG.1ABCD), and the BPDCN cell line PMDCN05, depending on the level of HLA-DP expression. The in vitro cytotoxicity of HLA-DP CARs was comparable to CD 19 CARs against JeKo-1 cells, similar to BCMA CARs against JJN-3 cells, and akin to CD123 CARs against PMDCN05 cells. HLA-DP and CD19 CARs killed 75% and 100% of JeKo-1 at 0.5:1 and 1:1 effector:tumor (E: T) ratio, respectively. Similarly, HLA-DP and BCMA CARs killed 90% and 100% of JJN-3 at 0.5:1 and 1:1 E: T, respectively. We did not detect cytotoxicity of HLA-DP CARs against HLA-DP KO JeKo-1 and JJN-3, demonstrating specificity.Additionally, cytotoxicity against 8 primary B-cell lymphomas derived from lymph nodes (2 follicular lymphomas (FL), 3 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphomas (MCL), and 1 Burkitt lymphoma (BL)) that expressed HLA-DP was assessed. We observed complete cytotoxicity versus FL and DLBCL, but not MCL and BL. Finally, we assessed the cytotoxicity against JeKo-1 cells using an in vivo mouse model. HLA-pan-DP CARsshowed a significantly longer survival benefit compared to empty CARs and no treatment (P < 0.05) and no difference to CD19 CARs (FIG. 2ABC).

[0064] Since HLA-DP is reported to be expressed on normal hematological cells such as B-cells, monocytes, macrophages, dendritic cells, as well as Schwann cells and endothelial cells, we assessed the cytotoxicity of HLA-pan-DP CARs against these cells. HLA-DP CARs killed B-cells, monocytes, macrophages, and dendritic cells, but not T-cells, NK-cells, and neutrophils. Using an xCelligence-based assay for alterations in target cell impedance upon loss of viability, no cytotoxic effects were found against Schwann cells (HE193, ipn02.3) and endothelial cells (HUVECs). We also assessed the influence of HLA-pan-DP CARs on hematopoiesis by colonyforming assays using methylcellulose-based medium. HLA-pan-DP CARs significantly reduced the number of colonies in CFU-M compared to empty CARs (P<0.001) but not in BFU-E, indicating a negative impact on B-cell and monocytic lineage hematopoiesis.

[0065] Conclusion: Anti -HLA-pan-DP CAR-T is promising for the treatment of various HLA-DP positive hematological malignancies such as B-cell lymphomas, MM, and particularly BPDCN, for which there are no effective treatments except for CD 123 targeted immunotherapy (SL-401). Due to expected suppression of normal hematopoiesis by HLA-pan-DP CAR-T, this approach would likely be best-implemented as a bridge to hematopoietic stem cell transplantation.Example 2Methods and materials for making of anti-HLA-pan-DP / CD3 bi-specific antibody

[0066] The bispecific antibody was converted from a scFv format to Fab format by humanization. Briefly, the variable light chain (VL) was fused to a human Constant Light chain kappa (CL), and similarly, the variable heavy chain (VH) was fused to Constant Heavy region 1 (CHI). To generate a bispecific antibody targeting T cell, the anti-CD3 clone okt3 was derived from Blinatumomab and fused to the C-terminus of CL. (FIG. 3).

[0067] To express the bi-specific antibody, corresponding plasmids were co-transfected in Expi293 cells using the FectoPRO transfection reagent, following the manufacturer’s protocol. At 5 days post-transfection, cells were harvested and centrifuged at 4,000 x g for 20 minutes, then supernatants were collected and passed through a 0.45μm syringe filter. Proteins were then purified on Capture select™ CH1-XL Pre-packed Column (Thermo Fisher), eluted with 0.1Macetic acid, neutralized with 1M Tris pH 11, and then buffer-exchanged into 1X PBS. Protein concentrations were estimated based on protein absorbance at 280nm with a Nanodrop spectrophotometer (Thermo Fisher).

[0068] To check for bispecific antibody binding on PBMCs, two donor PBMCs (dn3 and dn4) were used for staining. Briefly, 2M PBMCs or negative control HL60 cells were resuspended in ~0.2mL flow buffer (PBS Ca / Mg-free, 1% BSA, 0.5mM EDTA) (density ~10M cells / mL) and were incubated with the bispecific antibody at 4C for one hour, washed twice with flow buffer, and incubated with secondary antibody (Alexa Fluor® 488 AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, F(ab')2 fragment specific) for 30 minutes on ice and washed twice. PBMCs were then stained for surface expression using commercial antibodies CD3-PerCP-(UCHT1) and CD56 APC, and washed twice. Cells were resuspended in 100 pL of PBS (no BSA) and analyzed by flow cytometry. The bispecific antibody robustly stained the T-cell population in PBMC (CD3+ / CD56-) from two donors but not HL60 cell lines (FIG. 4).The CDRs of the bispecific antibody was the same as the CAR-T construct in SEQ ID NO: 1 and SEQ ID NO: 5 as shown below:anti-DP(murine VH)-CH1 (human) OIOLVQSGPELKKPGETVRISCKASGYIFTTAGMOWVOKMPGKGLKWIGWINTHSGEPK YAEDFKGRFALSLETSASTAYLOISNLKNEDTATYFCAREGLRRPYWYFDVWGAGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:22) anti-DP(murine VL)-CL(human kappa)-anti-CD3(okt3 VH-VL derived from BLINATUMOMAB) DIQMTOSPSSLSASLGGKVTITCKANQDINKYITWYOHKPGKGPRLLIHYTSTLOPGIPSR FSGSGSGRDYSFSISNLEPEDVATYFCLQYPNLPTFGGGTKLEIKRAVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSDIK£22XGAE£A / fPG yEAMX CKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKA TLTTDKSSSTA Y MQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLT QSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK( E ID NO 23)Binding of anti-HLA-DP BsAb

[0069] The anti-HLA-DP / CD3 bispecific antibody showed binding to all HLA-DP alleles tested.T cell-mediated cytotoxicity and T-cell activation using a multiple myeloma cell line and healthy donor T-cells

[0070] A multiple myeloma cell line (JJN-3) was incubated in 0.5 mM CellTrace CFSE (Life Technologies) for 8 minutes, washed twice, and resuspended at 105cells / mL in complete medium containing 2 mg / mL of Fc block. An assay was established by using 90 μL (2 x 104) of target cells and 90 μL (1 x 105) of fresh normal PBMC cells along with 20 pL of lOx concentration of antibody dilutions, and they were incubated at 37°C with 5% carbon dioxide for 48 hours in a 96-well U-bottom plate. After the incubation, plates were spun at 1500 rpm for 3 minutes, and the supernatants were collected for cytokine analysis. The cell pellets were stained with Live / Dead solution (ThermoFisher). Samples were analyzed by using BD FACS Lyrics. For cytotoxicity, the percentage of dead cells in CFSE+ cells was measured. The E (T-cell): T (JJN-3) ratio was 5:1. Teclistamab (Bispecific antibody against BCMA and human CD3; positive control), anti-βGal-hCD3 (Bispecific antibody against β-galactosidase and human CD3;Negative control, InvivoGen) and anti-HLA-DP-hCD3 BsAbs were used. FIG. 5 shows cytotoxicity of BsAbs against JJN-3. The anti-HLA-DP-hCD3 BsAb showed higher cytotoxicity against JJN-3 than teclistamab and anti-βGal-hCD3 BsAbs.

[0071] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.SEQUENCES SEQ ID NO: 1Heavy chain variable region (HCDRs are underlined):OIOLVQSGPELKKPGETVRISCKASGYIFTTAGMOWVOKMPGKGLKWIGWINTHSGEPK YAEDFKGRFALSLETSASTAYLOISNLKNEDTATYFCAREGLRRPYWYFDVWGAGTTV TVSS SEQ ID NO: 2HCDR1: GYIFTTAGSEQ ID NO: 3HCDR2: INTHSGEPSEQ ID NO: 4HCDR3: AREGLRRPYWYFDVSEQ ID NO: 5Light chain variable region (LCDRs are underlined):DIQMTOSPSSLSASLGGKVTITCKANQDINKYITWYQHKPGKGPRLLIHYTSTLOPGIPSR FSGSGSGRDYSFSISNLEPEDVATYFCLQYDNLPTFGGGTKLEIKR SEQ ID NO: 6LCDR1: QDINKYISEQ ID NO: 7LCDR2: YTSTSEQ ID NO: 8LCDR3: LQYDNLPTSEQ ID NO: 9EXEMPLARY HLA DP alpha protein (encoded by the DPA1 *01:03:01:01 allele) MRPEDRMFHIRAVILRALSLAFLLSLRGAGAIKADHVSTYAAFVQTHRPTGEFMFEFDE DEMFYVDLDKKETVWHLEEFGQAFSFEAQGGLANIAILNNNLNTLIQRSNHTQATNDPP EVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVTEGVAESLFLPRTDYSFHK FHYLTFVPSAEDFYDCRVEHWGLDQPLLKHWEAQEPIQMPETTETVLCALGLVLGLVGI IVGTVLIIKSLRSGHDPRAQGTL SEQ ID NO:10: Exemplary scFv amino acid sequence QIQLVQSGPELKKPGETVRISCKASGYIFTTAGMQWVQKMPGKGLKWIGWINTHSGEPK YAEDFKGRFALSLETSASTAYLQISNLKNEDTATYFCAREGLRRPYWYFDVWGAGTTV TVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGGKVTITCKANQDINKYITWYQHKP GKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDVATYFCLQYDNLPTFGGGT KLEIKR SEQ ID NO: 11: Exemplary amino acid linker between Vh and VL amino acid sequences in SEQ ID NO: 10GGGGSGGGGSGGGGS SEQ ID NO: 12, Exemplary CAR, with separate portions listed:SEQ ID NO:13; Signal peptideMALPVTALLLPLALLLHAARP SEQ ID NO:14; Myc TacEQKLISEEDLEEDLSEQ TD NO: 15; scFv_VHVLHeavy chain, Spacer (underlined), Light chain QIQLVQSGPELKKPGETVRTSCKASGYIFTTAGMQWVQKMPGKGLKWIGWTNTHSGEPK YAEDFKGRFALSLETSASTAYLQISNLKNEDTATYFCAREGLRRPYWYFDVWGAGTTV TVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASLGGKVTITCKANODINKYITWYQHKP GKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDVATYFCLQYDNLPTFGGGT KLEIKR SEQ ID NO: 16; IgG4 (EQ) Spacer ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS1EKT1S KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:17; CD28 TransmembraneFWVLVVVGGVLACYSLLVTVAFTIFWV SEQ ID NO:18; CD28 Co-Stim domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:19; CD3z RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRThe sequence between CD3z and 4XGS LinkerS SEQ ID NO:20; 4XGS LinkerGSGSGSGS SEQ ID NO:21; GFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMD ELYK*SEQ ID NO:22; Exemplary anti-DP(murine VH)-CH1 (human) OIOLVOSGPELKKPGETVRISCKASGYIFTTAGMOWVOKMPGKGLKWIGWINTHSGEPK YAEDFKGRFALSLETSASTAYLOISNLKNEDTATYFCAREGLRRPYWYFDVWGAGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQ S SGL YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVSEQ TD NO:23; Exemplary anti-DP(murine VL)-CL(human kappa)-anti-CD3(okt3 VH-VL derived from BLINATUMOMAB). Bold and italicized amino acids are anti-CD3(okt3 VH-VL derived from BLINATUMOMAB) DIQMTOSPSSLSASLGGKVTITCKANQDINKYITWYOHKPGKGPRLLIHYTSTLOPGIPSR FSGSGSGRDYSFSISNLEPEDVATYFCLQYPNLPTFGGGTKLEIKRAVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSDI DIKLQQSGAELARPGASVKMS CKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY MQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLT QSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

Claims

WHAT IS CLAIMED IS:

1. A human cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular binding domain that specifically binds to Human Leukocyte Antigen (HLA)-DP, wherein the extracellular binding domain is linked to a transmembrane domain and optionally at least one intracellular signaling domain.

2. The human cell of claim 1, wherein the human cell is a T-cell, natural killer (NK) cell, lymphocyte or a macrophage.

3. The human cell of claim 1 or 2, wherein the extracellular binding domain comprises a heavy chain variable region and a light chain variable region from an antibody that binds to HLA-DP.

4. The human cell of any one of claims 1-3, wherein(a) the heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HDR2, wherein HCDR1 comprises SEQ ID NO: 2, HCDR2 comprises SEQ ID NO:3 and HCDR3 comprises SEQ ID NO:4; and / or(b) the light chain variable region comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LDR2, wherein LCDR1 comprises SEQ ID NO: 6, LCDR2 comprises SEQ ID NO:7 and LCDR3 comprises SEQ ID NO:8.

5. The human cell of claim 4, wherein the heavy chain variable region comprises an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO: 1 and / or the light chain variable region comprises an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO:5.

6. The human cell of any one of claims 1-7, wherein the extracellular binding domain is a scfv.

7. The human cell of any one of claims 1-6, wherein the intracellular signaling domain is an immunoreceptor tyrosine-based activation motif (ITAM).

8. The human cell of any one of claims 1-7, wherein the CAR comprises two or more intracellular signaling domains.

9. The human cell of any one of claims 1-8, wherein the intracellular signaling domain is selected from the group consisting of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAPlO and DAP12.

10. The human cell of any one of claims 1-9, wherein at least one intercellular signaling domain is a co-stimulatory domain.

11. The human cell of claim 10, wherein the co-stimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

12. A nucleic acid comprising a coding sequence encoding the CAR of any of claims 1-11.

13. An expression cassette comprising a promoter operably linked to the coding sequence of claim 12.

14. A vector comprising the nucleic acid of claim 12 or the expression cassette of claim 13.

15. The vector of claim 14, wherein the vector is a viral vector.

16. A method of making a cell expressing a chimeric antigen receptor (CAR), the method comprising,introducing the nucleic acid of claim 12, or the expression cassette of claim 13, or the vector of any one of claims 14-15 into the cell such that the cell expresses the CAR.

17. The method of claim 16, wherein the introducing is in vitro or ex vivo.

18. The method of claim 16, further comprising, before the introducing, isolating the cell from a human.

19. The method of any one of claims 16-18, wherein the cell is a T-cell, natural killer (NK) cell, lymphocyte or a macrophage.

20. A method of killing a cancer cell expressing HLA-DP in a human, the method comprising administering the human cell of any one of claims 1-11 to the human, wherein at least one cancer in the human is killed.

21. The method of claim 20, further comprising before the administering, isolating a cell from the human; andexpressing the CAR in the cell.

22. The method of claim 20 or 21, wherein the human has received radiation therapy to kill hematopoietic stem cells prior to the administering.

23. The method of claim 22, wherein the hematopoietic stem cells are administered to the human before, with or after the administering of the human cell.

24. The method of claim 23, wherein the hematopoietic stem cells administered to the human are autologous or allogeneic to the human.

25. The method of any one of claims 20-24, wherein the cancer is lymphoma.

26. The method of claim 25, wherein the lymphoma is a B-cell lymphoma.

27. A bispecific antibody that specifically binds to Human Leukocyte Antigen (HLA)-DP and a second target.

28. The bispecific antibody of claim 27, wherein the second target is CD329. The bispecific antibody of claim 28, wherein the bispecific antibody comprises (i) a first polypeptide comprising an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO: 22 and (ii) a second polypeptide comprising an amino acid sequence at least 90, 95, 98, 99 or 100% identical to SEQ ID NO: 23.

30. A nucleic acid comprising a coding sequence encoding the first polypeptide or the second polypeptide or both the first polypeptide and the second polypeptides of claim 29.

31. An expression cassette comprising a promoter operably linked to the coding sequence of claim 30.

32. A vector comprising the nucleic acid of claim 30 or the expression cassette of claim 31.

33. The vector of claim 32, wherein the vector is a viral vector.

34. A method of making a cell expressing a bispecific antibody that specifically binds to Human Leukocyte Antigen (HLA)-DP and CD3, the method comprising, introducing the nucleic acid of claim 30, or the expression cassette of claim 31, or the vector of any one of claims 32-33 into the cell such that the cell expresses the bispecific antibody.

35. A method of killing a cancer cell expressing HLA-DP in a human, the method comprising administering the bispecific antibody of any one of claims 27-29 to the human, wherein at least one cancer in the human is killed.

36. The method of claim 35, wherein the cancer is lymphoma.

37. The method of claim 36, wherein the lymphoma is a B-cell lymphoma.

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