Anti-CD79 chimeric antigen receptors, CAR-T cells, and their use

Abstract

The present disclosure provides chimeric antigen receptors (CARs) that specifically target the cluster of differentiation 79b protein (CD79b) for the treatment of cancer, and immunoresponsive cells comprising such CARs.

Description

Technical Field

[0001] (Sequence Listing) This application includes a sequence listing that has been electronically filed in ASCII format and is hereby incorporated by reference in its entirety. The above ASCII copy was created on November 9, 2020, has the name JBI6171WOPCT1_SL.txt, and is 488,479 bytes in size.

[0002] (Field of the Invention) The present invention relates to a chimeric antigen receptor (CAR) targeting CD79b, comprising a single-chain variable fragment targeting CD79b, and a genetically engineered immune cell targeting CD79b that expresses the CAR. Also provided are a nucleic acid encoding the CAR and an expression vector, a recombinant cell containing the vector, and a composition comprising a genetically engineered immune cell expressing a CAR targeting CD79b. Methods for producing the CAR and the genetically engineered immune cells, and methods for using the genetically engineered immune cells for treating conditions including cancer are also provided.

Background Art

[0003] In T cell therapy, genetically modified and isolated T cells are utilized to enhance specificity for specific tumor-associated antigens. The genetic modification involves the expression of a chimeric antigen receptor (CAR) or an exogenous T cell receptor and can provide new antigen specificity on the T cells. T cells expressing a chimeric antigen receptor (CAR-T cells) can induce tumor immunoreactivity. There is a need for better cancer therapies using CAR-T cells.

[0004] Non-Hodgkin lymphoma (NHL) accounts for approximately 4% of all cancers. Despite improvements in available therapies, relapsed / refractory (r / r) NHL is characterized by a uniformly poor prognosis. Adoptive immunotherapy using T cells genetically engineered to express chimeric antigen receptors (CARs) has shown promising results in the treatment of CD19-positive B-cell malignancies. However, even with an initial overall response rate of approximately 60–80%, only 40% of patients achieve long-term complete remission [1, 2]. Clinical data are now emerging reporting disease relapse due to CD19 antigen loss in both acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL) patients, highlighting an unaddressed clinical need for novel surface antigen targeting [2, 4].

[0005] B cells (B lymphocytes) are central components of adaptive immunity, responding to several different pathogens by producing antibodies, acting as antigen-presenting cells, secreting cytokines, and developing into memory B cells after activation.[5] B cells circulate in the blood and lymphatic system. In lymphoid organs, they can encounter their congener antigens and, with additional signaling from T helper cells, differentiate into effector plasma cells. These cells circulate in the blood and secrete specific antibodies that target and eliminate antigens or pathogens.[6]

[0006] To detect antigens or pathogens, B cells possess a B cell receptor (BCR) on their cell surface, which is a multicomponent receptor consisting of transmembrane immunoglobulin molecules (mIg) and disulfide-bonded heterodimers of CD79a (Igα) and CD79b (Igβ). CD79b is highly expressed in a wide range of B-cell lymphomas. Its expression has been shown to be important for cancer cell survival in most DLBCL tumor models. Therefore, resistance to CD79b-targeted drugs due to antigen loss is unlikely, making it an attractive target for the development of novel immunotherapy approaches. Clinically, polatuzumab (Polivy®), an antibody-drug conjugate (ADC) molecule that targets CD79b, has recently been approved as a treatment for r / rDLBCL [7]. Polatuzumab treatment increased the complete response (CR) and duration of response (DOR) rates when combined with standard treatment (bendamustine and rituximab), and validated CD79b as a valuable clinical target.[8] [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need to develop CD79b-targeted CAR-T therapies. [Means for solving the problem]

[0008] Disclosed herein are chimeric antigen receptors (CARs), such as CARs targeting the cluster of differential antigen 79B protein (CD79b), cells containing CARs, vectors encoding CARs, such as recombinant expression vectors, and nucleic acid molecules encoding CARs, methods for producing CARs, compositions, polypeptides, proteins, nucleic acids, host cells, cell populations, and methods for treating disorders, such as cancer, using the disclosed CARs.

[0009] In one embodiment, a chimeric antigen receptor (CAR) is provided, comprising the following: (a) An extracellular domain containing scFv that specifically binds to the differentiated antigen group 79B protein (CD79b) antigen, (b) Transmembrane domain and (c) An intracellular signaling domain optionally containing at least one co-stimulatory domain.

[0010] In some embodiments, CAR is (d) CD8a-Hinge Region It further includes, The transmembrane domain contains a CD8a transmembrane region (CD8A-TM) polypeptide. The intracellular signaling domain includes a costimulatory domain containing a TNF receptor superfamily member 9 (CD137) component and a primary signaling domain containing a T cell surface glycoprotein CD3ζ chain (CD3z) component.

[0011] In some embodiments, the CD8a hinge region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 38. The transmembrane domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 39, and / or The intracellular signaling domain includes a co-stimulatory domain having an amino acid sequence at least 90% identical to SEQ ID NO: 40, and a primary signaling domain having an amino acid sequence at least 90% identical to SEQ ID NO: 41.

[0012] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 208, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 209, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 210, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 216, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 217, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 218. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 228, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 217, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 229, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 232, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 233, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 234. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 238, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 239, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 240, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 242, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 244, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 248, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 249, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 250, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 253, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 254, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 255. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 257, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 258, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 259. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 263, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 264. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 268, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 269, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 270, or It comprises heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 274, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 275, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 276. A CAR is provided in which the extracellular antigen-binding domain binds to the CD79b antigen.

[0013] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, light chain CDR2 having the amino acid sequence of SEQ ID NO: 220, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 230, light chain CDR2 having the amino acid sequence of SEQ ID NO: 231, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 235, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 245, light chain CDR2 having the amino acid sequence of SEQ ID NO: 246, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 252. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 256. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 262. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 265, light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, light chain CDR2 having the amino acid sequence of SEQ ID NO: 272, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 273, or It comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 278. A CAR is provided in which the extracellular antigen-binding domain binds to the CD79b antigen.

[0014] In some embodiments, the extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, light chain CDR2 having the amino acid sequence of SEQ ID NO: 220, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 230, light chain CDR2 having the amino acid sequence of SEQ ID NO: 231, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 235, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 245, light chain CDR2 having the amino acid sequence of SEQ ID NO: 246, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 252. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 256. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 262. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 265, light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, light chain CDR2 having the amino acid sequence of SEQ ID NO: 272, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 273, or The present invention further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 278. In another embodiment, a chimeric antigen receptor (CAR) is provided comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 208, 216, 222, 228, 232, 238, 242, 248, 253, 257, 263, 268, or 274, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 209, 217, 223, 233, 239, 243, 249, 254, 258, 269, or 275, and SEQ ID NO: 210, 218, 224, 229, 234, 240, 244, This includes a heavy chain CDR3 having the amino acid sequence 250, 255, 259, 264, 270, or 276; a light chain CDR1 having the amino acid sequence 211, 214, 215, 219, 225, 230, 235, 241, 245, 251, 260, 265, 271, or 277; a light chain CDR2 having the amino acid sequence 212, 220, 226, 231, 236, 246, 261, 266, or 272; and a light chain CDR3 having the amino acid sequence 213, 221, 227, 237, 247, 252, 256, 262, 267, 273, or 278.

[0015] In some embodiments, the extracellular antigen-binding domain is a) Sequence numbers 208, 209, 210, 211, 212, and 213 respectively, b) Sequence numbers 208, 209, 210, 214, 212, and 213 respectively, c) Sequence numbers 208, 209, 210, 215, 212, and 213 respectively, d) Sequence numbers 216, 217, 218, 219, 220, and 221, respectively e) Sequence numbers 222, 223, 224, 225, 226, and 227, respectively f) Sequence numbers 228, 217, 229, 230, 231, and 221 respectively, g) Sequence numbers 232, 233, 234, 235, 236, and 237, respectively h) Sequence numbers 238, 239, 240, 241, 226, and 227 respectively, i) Sequence numbers 242, 243, 244, 245, 246, and 247, respectively j) Sequence numbers 248, 249, 250, 251, 236, and 252, respectively k) Sequence numbers 253, 254, 255, 251, 236, and 256 respectively, l) Sequence numbers 257, 258, 259, 260, 261, and 262, respectively m) Sequence numbers 263, 243, 264, 265, 266, and 267 respectively, n) Sequence numbers 268, 269, 270, 271, 272, and 273, respectively, or o) comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, each having the amino acid sequences of SEQ ID NOs. 274, 275, 276, 277, 266, and 278, respectively.

[0016] In some embodiments, the extracellular antigen-binding domain is A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 22. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 23. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 27, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 30, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 31, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 34. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 35. A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 17, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, or It includes a heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 36.

[0017] In some embodiments, the extracellular antigen-binding domain is A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 4, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 23, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 25, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 27, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 28, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 29, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 33, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 35, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 33, or It includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36.

[0018] In some embodiments, the extracellular antigen-binding domain is It includes a heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 32.

[0019] In some embodiments, the extracellular antigen-binding domain is It includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32.

[0020] In some embodiments, the extracellular antigen-binding domain includes a single-chain variable fragment (scFv). In some embodiments, the scFv includes a linker polypeptide between the light chain variable region (VL) and the heavy chain variable region (VH). In some embodiments, the linker polypeptide includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 42. In some embodiments, the linker polypeptide includes the amino acid sequence of SEQ ID NO: 42. In some embodiments, the scFv includes an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 75 to 118. In some embodiments, the scFv includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 75 to 118. In some embodiments, the extracellular antigen-binding domain includes a signal polypeptide. In some embodiments, the signal polypeptide includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 37. In some embodiments, the signal polypeptide includes the amino acid sequence of SEQ ID NO: 37.

[0021] In some embodiments, the intracellular signaling domain includes polypeptide components selected from the group consisting of TNF receptor superfamily member 9 (CD137) components, T cell surface glycoprotein CD3ζ chain (CD3z) components, differentiation cluster (CD27) components, differentiation cluster superfamily member components, and combinations thereof. In some embodiments, the CD137 component includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 40. In some embodiments, the CD137 component includes the amino acid sequence of SEQ ID NO: 40. In some embodiments, the CD3z component includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 41. In some embodiments, the CD3z component includes the amino acid sequence of SEQ ID NO: 41. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 163. In some embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 163. In some embodiments, the transmembrane domain includes a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 39. In some embodiments, the CD8a-TM polypeptide contains the amino acid sequence of SEQ ID NO: 39.

[0022] In various embodiments, the CAR further includes a hinge region that links the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 38. In some embodiments, the CD8a hinge region includes the amino acid sequence of SEQ ID NO: 38.

[0023] In some embodiments, the extracellular antigen-binding domain includes an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 119-162.

[0024] In some embodiments, the CAR includes an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 164-207.

[0025] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes heavy chain complementarity determination region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 257, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 258, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 259.

[0026] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes a light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 262.

[0027] In some embodiments, the extracellular antigen-binding domain may include heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, each having the amino acid sequences of SEQ ID NOs. 257, 258, 259, 260, 261, and 262, respectively.

[0028] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes a heavy chain variable region (VH) having an amino acid sequence at least 90% identical to SEQ ID NO: 14, and a light chain variable region (VL) having an amino acid sequence at least 90% identical to SEQ ID NO: 32, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0029] In another embodiment, a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 32, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0030] In various embodiments, the extracellular antigen-binding domain comprises a single-strand variable fragment (scFv), which comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). The scFv may also contain a linker polypeptide between the light-chain variable region (VH) and the heavy-chain variable region (VL). In certain embodiments, the linker polypeptide may contain an amino acid sequence that is at least 90% identical to SEQ ID NO: 42. In certain embodiments, the linker polypeptide may contain the amino acid sequence of SEQ ID NO: 42.

[0031] In certain embodiments, scFv may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 113.

[0032] In some embodiments, the extracellular antigen-binding domain may include a signal polypeptide. In such embodiments, the signal polypeptide may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 37.

[0033] In some embodiments, the intracellular signaling domain may include polypeptide components selected from the group consisting of TNF receptor superfamily member 9 (CD137) components, T cell surface glycoprotein CD3ζ chain (CD3z) components, differentiation cluster (CD27) components, differentiation cluster superfamily member components, and combinations thereof. As an unrestricted example, the CD137 component may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 40. The CD3z component may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 41, and the intracellular signaling domain may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 163. As another unrestricted example, the CD137 component may include the amino acid sequence of SEQ ID NO: 40. The CD3z component may include the amino acid sequence of SEQ ID NO: 41, and the intracellular signaling domain may include the amino acid sequence of SEQ ID NO: 163.

[0034] In certain embodiments, the intracellular signaling domain may include an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 163.

[0035] In some embodiments, the transmembrane domain may comprise a CD8a transmembrane region (CD8a-TM) polypeptide. In such embodiments, the CD8a-TM polypeptide may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 39. In such embodiments, the CD8a-TM polypeptide may comprise the amino acid sequence of SEQ ID NO: 39.

[0036] In some embodiments, the CARs disclosed herein may further include a hinge region that links the transmembrane domain to an extracellular antigen-binding domain. In certain embodiments, the hinge region may be a CD8a hinge region. In some embodiments, the CD8a hinge region may include an amino acid sequence that is at least 90% identical to SEQ ID NO: 38. In such embodiments, the CD8a hinge region may include the amino acid sequence of SEQ ID NO: 38.

[0037] In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 157.

[0038] In some embodiments, the CARs disclosed herein may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 202. In some embodiments, the CARs disclosed herein may comprise the amino acid sequence of SEQ ID NO: 202. In another embodiment, isolated lymphocytes expressing any of the above CARs are provided. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the lymphocytes are natural killer (NK) cells.

[0039] Also provided are isolated nucleic acid molecules encoding any of the above CARs. Vectors containing nucleic acid molecules are also provided. Furthermore, cells expressing nucleic acid molecules are also provided.

[0040] Furthermore, pharmaceutical compositions are also provided, comprising an effective amount of any of the above-mentioned lymphocytes and a pharmaceutically acceptable excipient.

[0041] In another embodiment, a method is provided for treating a subject having cancer. The method comprises administering a therapeutically effective amount of any of the above lymphocytes or the above pharmaceutical composition to a subject in need thereof, thereby inducing the lymphocytes to kill cancer cells in the subject. In some embodiments, the cancer is B-cell lymphoma. In some embodiments, the cancer is non-Hodgkin lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasmacytoma.

[0042] In another embodiment, a method for targeted death of cancer cells is provided, the method comprising contacting the cancer cells with any of the lymphocytes described above, thereby inducing the death of the cancer cells by the lymphocytes. In some embodiments, the cancer cells are malignant B cells. In some embodiments, the cancer cells are cells of non-Hodgkin lymphoma. In some embodiments, the cancer cells are cells of diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasmacytoma.

[0043] In another embodiment, a method for detecting the presence of cancer in a subject, (a) Forming a CAR-cell complex by contacting a cell sample obtained from the subject with any of the above CARs, (b) A method is provided which includes detecting a complex (where the detection of the complex indicates the presence of cancer in the subject). [Brief explanation of the drawing]

[0044] The foregoing will become clear from a more detailed description of the exemplary embodiments shown in the accompanying drawings below.

[0045] This patent application document includes at least one color drawing. Copies of this patent application containing color drawings(s) will be provided by the Patent Office upon request and payment of the necessary fees. [Figure 1] This is a diagram of an exemplary CD79b CAR molecular structure. CD79b scFv was cloned sequentially using the CD8a hinge / transmembrane domain, the CD137 intracellular domain, and the CD3ζ intracellular domain. [Figure 2A] This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2B]This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2C] This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2D]This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2E] This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2F]This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2G] This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2H]This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 2I] This document describes the generation of primary human CAR-T cells expressing CD79b CAR. Primary human T cells were isolated by negative selection and stimulated with TransAct (Miltenyi) in TexMACS medium (Figures 2A-2C, 2G-2I) or with anti-CD3 / anti-CD28 beads (Dynabeads, Invitrogen) in Optimizer medium (Figures 2D-2F). In both cases, the medium was supplemented with 100 U / mL of IL-2 (Miltenyi). Cells were transduced with a lentiviral vector encoding the CAR construct 24 hours after stimulation and cultured for 12-14 days. The medium and cytokines were refreshed every 2-4 days. CD79b CAR expression was quantified via flow cytometry using a recombinant human CD79b extracellular domain fused to the AF647 protein. The frequency of CAR+ cells is shown. In Figures 2A-AC and 2E, all SN8(CD9W) structures are in LH orientation. [Figure 3A]This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 3B] This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 3C]This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 3D] This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 3E]This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 3F] This study demonstrates that CD79b CAR exhibits cytotoxicity against CD79b+ tumor cell lines. CD79b CAR was co-cultured with the indicated target cell lines for 16–20 hours at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Tumor lysis was assessed by flow cytometry after viability staining (Figure 3A) or by the disappearance of tumor cell luciferase signaling using the Promega BrightGlo kit (Figures 3B–3F), according to the manufacturer's instructions. In each case, the lysis percentage was calculated relative to the tumor alone. Dose-dependent antitumor activity of the CAR in CD79b+ target cells was observed, and no background-level lysis against the CD79bneg target was seen. In Figures 3A–3D, all SN8 (CD9W) constructs are LH-oriented. In Figures 3E–3F, HL or LH orientation is indicated by the first letter. [Figure 4A]This study demonstrates that CD79b CAR secretes cytokines in response to stimulation by antigen-positive tumor cells. Supernatants of co-cultures prepared as shown in Figure 3 were collected 16–20 hours after preparation, and cytokines were quantified using the MSD kit (Meso Scale Diagnostics) according to the manufacturer's protocol. CD79b CAR exhibited antigen-dependent cytokine production. In Figures 4A–4C, all SN8 (CD9W) constructs are LH-oriented. In Figure 4D, HL or LH orientation is indicated by the first letter. [Figure 4B] This study demonstrates that CD79b CAR secretes cytokines in response to stimulation by antigen-positive tumor cells. Supernatants of co-cultures prepared as shown in Figure 3 were collected 16–20 hours after preparation, and cytokines were quantified using the MSD kit (Meso Scale Diagnostics) according to the manufacturer's protocol. CD79b CAR exhibited antigen-dependent cytokine production. In Figures 4A–4C, all SN8 (CD9W) constructs are LH-oriented. In Figure 4D, HL or LH orientation is indicated by the first letter. [Figure 4C] This study demonstrates that CD79b CAR secretes cytokines in response to stimulation by antigen-positive tumor cells. Supernatants of co-cultures prepared as shown in Figure 3 were collected 16–20 hours after preparation, and cytokines were quantified using the MSD kit (Meso Scale Diagnostics) according to the manufacturer's protocol. CD79b CAR exhibited antigen-dependent cytokine production. In Figures 4A–4C, all SN8 (CD9W) constructs are LH-oriented. In Figure 4D, HL or LH orientation is indicated by the first letter. [Figure 4D]This study demonstrates that CD79b CAR secretes cytokines in response to stimulation by antigen-positive tumor cells. Supernatants of co-cultures prepared as shown in Figure 3 were collected 16–20 hours after preparation, and cytokines were quantified using the MSD kit (Meso Scale Diagnostics) according to the manufacturer's protocol. CD79b CAR exhibited antigen-dependent cytokine production. In Figures 4A–4C, all SN8 (CD9W) constructs are LH-oriented. In Figure 4D, HL or LH orientation is indicated by the first letter. [Figure 5A] This study demonstrates that CD79b CARs specifically proliferate in the presence of the antigen. CD79b CARs were labeled with CellTraceViolet (CTV) according to the manufacturer's instructions (Invitrogen). Labeled CAR-T cells were co-cultured with the designated target at a 1:2 E:T ratio for 4–5 days, and then stained with a CAR detection reagent (recombinant CD79b-AF647). Next, the proliferation of CAR+ cells was analyzed by flow cytometry. The plots show the CTV dilution of CAR+ cells. CD79b CARs specifically showed CTV dilution (proliferation) when stimulated with the CD79b tumor line, but not with the antigen-negative target line. In Figures 5A–5C, all SN8 (CD9W) constructs are LH-oriented. In Figure 5C, HL or LH orientation is indicated by the first letter. [Figure 5B] This study demonstrates that CD79b CARs specifically proliferate in the presence of the antigen. CD79b CARs were labeled with CellTraceViolet (CTV) according to the manufacturer's instructions (Invitrogen). Labeled CAR-T cells were co-cultured with the designated target at a 1:2 E:T ratio for 4–5 days, and then stained with a CAR detection reagent (recombinant CD79b-AF647). Next, the proliferation of CAR+ cells was analyzed by flow cytometry. The plots show the CTV dilution of CAR+ cells. CD79b CARs specifically showed CTV dilution (proliferation) when stimulated with the CD79b tumor line, but not with the antigen-negative target line. In Figures 5A–5C, all SN8 (CD9W) constructs are LH-oriented. In Figure 5C, HL or LH orientation is indicated by the first letter. [Figure 5C] This study demonstrates that CD79b CARs specifically proliferate in the presence of the antigen. CD79b CARs were labeled with CellTraceViolet (CTV) according to the manufacturer's instructions (Invitrogen). Labeled CAR-T cells were co-cultured with the designated target at a 1:2 E:T ratio for 4–5 days, and then stained with a CAR detection reagent (recombinant CD79b-AF647). Next, the proliferation of CAR+ cells was analyzed by flow cytometry. The plots show the CTV dilution of CAR+ cells. CD79b CARs specifically showed CTV dilution (proliferation) when stimulated with the CD79b tumor line, but not with the antigen-negative target line. In Figures 5A–5C, all SN8 (CD9W) constructs are LH-oriented. In Figure 5C, HL or LH orientation is indicated by the first letter. [Figure 6] This study demonstrates that CD79b CARs exhibit long-term cytotoxicity against CD79b+ tumor cell lines. CD79b CARs were co-cultured with the indicated RFP-expressing target cell lines for 4–7 days at the indicated effector:target (E:T) ratio (based on CAR+ frequency). Cell growth was tracked over time by imaging each well every 4 hours and calculating the total RFP+ area / well using IncuCyte® technology. Growth inhibition was calculated for each of the constructs tested. In Figure 6, all SN8(CD9W) constructs are LH-oriented. [Figure 7A] This study demonstrates that CD79b CAR-T cells eradicate CD79b+ tumors in vivo. 5 × 10⁵ CARNAVAL cells were subcutaneously transplanted into NOD / scid / IL-2Rg- / -(NSG) mice. CAR-T cells were intravenously injected when the mean group tumor volume reached approximately 50–100 mm³ / mouse (Figure 7A). Tumors (Figure 7B) and body weight (Figure 7C) were monitored twice weekly. [Figure 7B]This study demonstrates that CD79b CAR-T cells eradicate CD79b+ tumors in vivo. 5 × 10⁵ CARNAVAL cells were subcutaneously transplanted into NOD / scid / IL-2Rg- / -(NSG) mice. CAR-T cells were intravenously injected when the mean group tumor volume reached approximately 50–100 mm³ / mouse (Figure 7A). Tumors (Figure 7B) and body weight (Figure 7C) were monitored twice weekly. [Figure 7C] This study demonstrates that CD79b CAR-T cells eradicate CD79b+ tumors in vivo. 5 × 10⁵ CARNAVAL cells were subcutaneously transplanted into NOD / scid / IL-2Rg- / -(NSG) mice. CAR-T cells were intravenously injected when the mean group tumor volume reached approximately 50–100 mm³ / mouse (Figure 7A). Tumors (Figure 7B) and body weight (Figure 7C) were monitored twice weekly. [Figure 8A] This study demonstrates that basal cytokine levels in CD79b CAR-T transduced cells do not increase in the absence of antigen or cytokine stimulation. Previously generated CD79b CAR-T cells were seeded three times at 50,000 CAR+ cells per well. The total number of T cells per well was normalized under each condition by adding untransduced (UTD) cells. After overnight culture, the culture supernatant was collected, and cytokine levels were quantified using MSD (V Plex Proinflammation Panel 1 [Human] Kit). INFγ, IL-2, and TNFα levels detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells were plotted as mean (pg / mL) ± SEM for each donor. [Figure 8B]This study demonstrates that basal cytokine levels in CD79b CAR-T transduced cells do not increase in the absence of antigen or cytokine stimulation. Previously generated CD79b CAR-T cells were seeded three times at 50,000 CAR+ cells per well. The total number of T cells per well was normalized under each condition by adding untransduced (UTD) cells. After overnight culture, the culture supernatant was collected, and cytokine levels were quantified using MSD (V Plex Proinflammation Panel 1 [Human] Kit). INFγ, IL-2, and TNFα levels detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells were plotted as mean (pg / mL) ± SEM for each donor. [Figure 8C] This study demonstrates that basal cytokine levels in CD79b CAR-T transduced cells do not increase in the absence of antigen or cytokine stimulation. Previously generated CD79b CAR-T cells were seeded three times at 50,000 CAR+ cells per well. The total number of T cells per well was normalized under each condition by adding untransduced (UTD) cells. After overnight culture, the culture supernatant was collected, and cytokine levels were quantified using MSD (V Plex Proinflammation Panel 1 [Human] Kit). INFγ, IL-2, and TNFα levels detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells were plotted as mean (pg / mL) ± SEM for each donor. [Figure 9A]Abnormal basal proliferation or absence of activation was observed in 441-HL CAR-T cells in the absence of antigen or cytokine stimulation. A 5-day proliferation assay was performed with 441-HL CAR-T cells to screen for abnormal basal proliferation or activation. After labeling with Cell Trace® violet (CTV) dye (5 mM), CAR-T cells were diluted to 5 × 10⁵ viable CAR+ T cells per mL. Cells (100 μL) were then added to a 96-well round-bottom plate and grown for 5 days in the absence of target cells or cytokine stimulation. Target-positive cells (CARNAVAL) and target-negative cells (K562) were similarly seeded in a CAR+ effector:target (E:T) ratio of 1:1 and cultured under the same conditions. On day 5, cells were analyzed via flow cytometry gated with CD3, and then with CAR. The proliferation rate (Figure 9A) was determined for each of the five donors using CTV dye dilution (Pacific Blue channel) and CD71 marker expression. [Figure 9B] Abnormal basal proliferation or absence of activation was observed in 441-HL CAR-T cells in the absence of antigen or cytokine stimulation. A 5-day proliferation assay was performed with 441-HL CAR-T cells to screen for abnormal basal proliferation or activation. After labeling with Cell Trace® violet (CTV) dye (5 mM), CAR-T cells were diluted to 5 × 10⁵ viable CAR+ T cells per mL. Cells (100 μL) were then added to a 96-well round-bottom plate and grown for 5 days in the absence of target cells or cytokine stimulation. Target-positive cells (CARNAVAL) and target-negative cells (K562) were similarly seeded in a CAR+ effector:target (E:T) ratio of 1:1 and cultured under the same conditions. On day 5, cells were analyzed via flow cytometry gated with CD3, and then with CAR. The percentage of activated (Figure 9B) CAR-T cells was determined for each of the five donors using CTV dye dilution (Pacific Blue channel) and CD71 marker expression. [Figure 10A]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10B]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10C]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10D]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10E]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10F]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, viable cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). For the five donors, the integrated graphs for CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 10A-10C and 10D-10F, respectively. [Figure 10G]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10H]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10I]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10J]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10K]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10L]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10M]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10N]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10O]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10P]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10Q]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 10R]For all donors tested, 441-HL CAR-T cells demonstrated antigen-specific cancer cell killing in the presence of CD79b / CD19+ cells. Flow cytometry was used to test the efficacy of 441-HL CAR-T cells in a panel of five CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2, and JEKO-1) and five CD79b / CD19- cell lines (K562, HLY-1, SU-DHL-1, HL-60, and JURKAT E6.1). K562, engineered to express CD79b, was included as an additional control. After thawing and overnight standing, cells were counted, and the proportion of CAR+ cells was normalized per donor by adding untransduced cells. Next, normalized CAR-T cells were counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL, with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded into 96-well plates (100 μL per well). Target cell lines were harvested, counted, and resuspended at 4 million cells per mL. Subsequently, they were labeled with Cell Trace® Violet (CTV), diluted to 2 × 10⁵ viable cells per mL, and 100 μL of labeled cells were added to the 96-well plates containing CAR-T cells. After a 24-hour incubation period, the cells were stained with Fixable Viability Dye eFluor® 660. Tumor cell death was assessed by flow cytometry, gating forward and side scattering to identify cell populations. Next, surviving cells were identified using LIVE / DEAD, and finally, the number of surviving cancer cells in each well was evaluated using CTV+ tumor events. The cancer cell killing rate (%) was calculated by dividing the absolute number of surviving cancer cells in each E:T ratio by the average absolute number of surviving cancer cells in wells with an E:T ratio of 0:1, and multiplying by 100. The data were plotted as mean + / - SEM (three individual experiments). Plots showing the killing curves for each of the five donors are shown in Figures 10G-10L and 10M-10R for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 11A]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). The pooled graphs are shown in Figures 11A and 11B for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 11B]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). The pooled graphs are shown in Figures 11A and 11B for CD79b / CD19+ cells and CD79b / CD19- cells, respectively. [Figure 11C]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). Plots showing the individual killing curves for each donor against CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 11C-11D and 11E-11F, respectively. [Figure 11D]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). Plots showing the individual killing curves for each donor against CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 11C-11D and 11E-11F, respectively. [Figure 11E]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). Plots showing the individual killing curves for each donor against CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 11C-11D and 11E-11F, respectively. [Figure 11F]This report demonstrates the specific killing of antigen-positive cell lines by 441-HL CAR-T cells using IncuCyte® technology. Using IncuCyte® technology, the killing dynamics of 441-HL CAR-T cells were evaluated in a panel of two CD79b / CD19+ (HBL-1, OCI-LY-10) and two CD79b / CD19- (HLY-1, SU-DHL-1) mKATE2-expressing cell lines. After thawing and allowing to stand overnight, cells were counted, and the proportion of CAR+ cells was normalized for each donor by adding untransduced cells. The normalized CAR-T cells were then counted and resuspended at a concentration of 5 × 10⁵ CAR+ cells per mL with an initial CAR+E:T ratio of 2.5:1. A total of eight 2-fold dilutions were performed, and effector cells were seeded in 96-well plates (100 μL per well). Target cell lines were harvested and counted, resuspended at 1 × 10⁵ cells / mL, and seeded according to a plate layout (96-well plate, 100 μL per well). After mixing CAR-T cells with target cells, 80 μL from each well was dispensed twice into a 384-well plate. The co-culture was then placed in an IncuCyte® ZOOM live content imaging system, and images were automatically acquired (single image) every 4 hours for 4–6 days using a 4x objective lens in both phase and fluorescence channels. Target cell levels were quantified based on mKATE2 red fluorescent protein expression. To quantify cancer cell killing over time, the average area of ​​each replication was exported to GraphPad Prism, and area under the curve (AUC) values ​​were derived for each condition. After normalizing to an untreated control, the E:T ratio was plotted against the AUC value to determine the dose-response. For five donors, dose-response graphs were generated for both pooled values ​​(mean ± SEM) and individual values ​​(two independent experiments). Plots showing the individual killing curves for each donor against CD79b / CD19+ cells and CD79b / CD19- cells are shown in Figures 11C-11D and 11E-11F, respectively. [Figure 12A]This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of INFγ, IL-2, TNFα, and IL-6 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as integrated plots (mean ± SEM) across donors, are shown in Figures 12A-12D. [Figure 12B] This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of INFγ, IL-2, TNFα, and IL-6 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as integrated plots (mean ± SEM) across donors, are shown in Figures 12A-12D. [Figure 12C]This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of INFγ, IL-2, TNFα, and IL-6 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as integrated plots (mean ± SEM) across donors, are shown in Figures 12A-12D. [Figure 12D] This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of INFγ, IL-2, TNFα, and IL-6 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as integrated plots (mean ± SEM) across donors, are shown in Figures 12A-12D. [Figure 12E]This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of IL-4, IL-1β, IL10, and IL12p70 (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as merged (mean ± SEM) plots between donors, are shown in Figures 12E-12H. [Figure 12F] This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of IL-4, IL-1β, IL10, and IL12p70 (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as merged (mean ± SEM) plots between donors, are shown in Figures 12E-12H. [Figure 12G]This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of IL-4, IL-1β, IL10, and IL12p70 (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as merged (mean ± SEM) plots between donors, are shown in Figures 12E-12H. [Figure 12H] This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. Plots of IL-4, IL-1β, IL10, and IL12p70 (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as merged (mean ± SEM) plots between donors, are shown in Figures 12E-12H. [Figure 12I]This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. IL-13 and IL-8 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as aggregated plots (mean ± SEM) across donors, are shown in Figures 12I-12J. [Figure 12J] This study demonstrates antigen-specific production of pro-inflammatory cytokines by 441-HL CAR-TT cells. Supernatants from four CD79b / CD19+ cell lines (HBL-1, OCI-LY-10, CARNAVAL, WILL-2) and two CD79b / CD19- cell lines (HLY-1 and SU-DHL-1) were collected during flow cytometry-based killing assays and tested by Meso Scale Discovery (MSD). Data analysis was performed, and the results for each cytokine were plotted as individual values ​​for each of the five donors (two independent experiments) and as the mean values ​​for each treatment group. IL-13 and IL-8 levels (pg / mL) detected in the supernatants of 441-HL CAR-T, CD19 CAR-T, and UTD cells collected from each donor, as well as aggregated plots (mean ± SEM) across donors, are shown in Figures 12I-12J. [Figure 13A]This shows dose-dependent inhibition of tumor growth by CD79b CAR-T cells in a CARNAVAL xenograft model. Figure 13A shows a schematic diagram of the experimental paradigm used to investigate the in vivo efficacy of CD79b CAR-T cells in a CARNAVAL xenograft model. Logarithmically growing CARNAVAL cells (5 × 10⁵ cells) were subcutaneously transplanted into NOD / scid / IL-2Rg- / -(NSG) mice on day 0. When the mean group tumor volume reached approximately 50–100 mm³ / mouse (14 days post-transplant), the dose of CAR+ T cells described herein was administered intravenously. Tumor volume and body weight were recorded, and blood samples were collected periodically. [Figure 13B] This shows dose-dependent inhibition of tumor growth by CD79b CAR-T cells in the CARNAVAL xenograft model. The mean tumor volume (mm2 ± SEM) over the number of days after tumor transplantation is shown in Figure 13B. [Figure 13C] This shows dose-dependent inhibition of tumor growth by CD79b CAR-T cells in the CARNAVAL xenograft model. The mean percentage change in body weight (%±SEM) over the days after tumor transplantation is shown in Figure 13C. [Modes for carrying out the invention]

[0046] The following describes exemplary embodiments.

[0047] This disclosure provides chimeric antigen receptors (CARs) that target the differentiation antigen group 79B protein (CD79b), cells containing such CARs, and methods for treating cancer (e.g., B-cell lymphoma) using the CARs described herein.

[0048] The CAR of the present invention has antigen specificity for CD79b. The phrases “having antigen specificity” and “inducing an antigen-specific response,” as used herein, mean that the CAR can specifically bind to and immunologically recognize an antigen, and as a result, the binding of the CAR to the CD79b antigen induces an immune response. Methods for testing CARs for antigen specificity and their ability to recognize target cells are known in the art.

[0049] This disclosure also provides related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or their antigen-binding moieties, and pharmaceutical compositions relating to the CAR of the present invention.

[0050] Some aspects of the present invention are described below with reference to examples provided for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described in order to provide a complete understanding of the present invention. However, those skilled in the art will readily recognize that the present invention can be carried out without one or more of the specific details, or with other methods, protocols, reagents, cell lines, and animals. Since some actions may occur in a different order and / or in parallel with other actions or events, the present invention is not limited by the order of the illustrated actions or events. Furthermore, not all illustrated actions, steps, or events are required to carry out the methods of the present invention. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly used using conventional methods.

[0051] Unless otherwise defined, all technical terms, notations, and other scientific or specialized terms used herein are intended to have meanings that are commonly understood by those skilled in the art. In some cases, terms that have commonly understood meanings are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from the generally accepted understanding in the art. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with their meanings in the context of the relevant art and / or as otherwise defined herein.

[0052] definition Where a list is presented, please understand that, unless otherwise specified, each individual element of that list and all combinations of that list represent a distinct embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0053] The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. Where used herein, the articles "a," "an," and "the" should be understood to include multiple referents unless otherwise clearly indicated by the context.

[0054] The transitional phrases “comprising,” “consisting essentially of,” and “consisting” are intended to imply the generally accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the scope of the claims to specified materials or processes, and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with respect to the phrase “comprising” (or its equivalent) also provide embodiments that are described independently with respect to “consisting” and “consisting essentially of.”

[0055] The terms “about” or “approximately” include being within a statistically meaningful range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the specific system under study and can be readily understood by those skilled in the art.

[0056] "Activation" or "stimulation" refers to the induction of a change in the biological state of a cell (e.g., T cells and NK cells) that causes the cell to express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be induced by a primary stimulus signal. Co-stimulatory signals can amplify the magnitude of the primary signal and suppress cell death after initial stimulation, resulting in a more durable activated state and, consequently, higher cytotoxicity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, induces the proliferation of T cells and / or NK cells, as well as the upregulation or downregulation of key molecules.

[0057] "Bispecificity" refers to a molecule (e.g., an antibody) that specifically binds to two different antigens within the same antigen, or to two different epitopes. Bispecific molecules may cross-react to other related antigens, such as those from humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (homlogs), or they may bind to epitopes shared between two or more different antigens.

[0058] The terms “chimeric antigen receptor” or “CAR,” as used herein, are defined as cell surface receptors comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are combinations not found together in a single protein in nature. This includes, in particular, receptors in which the extracellular domain and intracellular signaling domain are not found together in a single receptor protein in nature. The chimeric antigen receptors of the present invention are primarily intended for use in lymphocytes such as T cells and natural killer (NK) cells.

[0059] The term “coding” refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a specific sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a specific sequence of amino acids, and the biological properties that arise therefrom, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in a cell or other biological system, if the transcription and translation of mRNA corresponding to a gene produces a protein, then that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand used as a template for the transcription of a gene or cDNA, can be referred to as coding for a protein or other product of that gene or cDNA.

[0060] Unless otherwise stated, “nucleotide sequences encoding an amino acid sequence” includes all nucleotide sequences that are degenerate of each other, and all nucleotide sequences encoding the same amino acid sequence. The phrase “nucleotide sequences encoding a protein or RNA” may also include introns to the extent that in some cases the nucleotide sequence encoding that protein may contain introns.

[0061] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression control sequence manipulably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by a host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0062] "Heterogeneous" refers to two or more polynucleotides or two or more polypeptides that are not found in nature in the same relationship to one another.

[0063] "Heterogeneic polynucleotide" refers to a polynucleotide that does not exist in nature and encodes two or more neoantigens as described herein.

[0064] "Heterogeneous polypeptide" refers to a polypeptide that does not exist in nature and contains two or more neoantigen polypeptides as described herein.

[0065] A "host cell" refers to any cell containing heterologous nucleic acids. An example of heterologous nucleic acid is a vector (e.g., an expression vector).

[0066] The terms “T cell” and “T lymphocyte” are interchangeable and are used synonymously herein. As used herein, T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cell may be a T helper (Th) cell, e.g., a T helper 1 (Th1) or T helper 2 (Th2) cell. T cell may be a helper T cell (HTL, CD4+ T cell), a CD4+ T cell, a cytotoxic T cell (CTL, CD8+ T cell), a tumor-infiltrating cytotoxic T cell (TIL, CD8+ T cell), a CD4+CD8+ T cell, or any other subset of T cell. Other exemplary populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. Furthermore, "NKT cells" are also included, which refer to a special population of T cells that not only express semi-invariant αβ T cell receptors but also various molecular markers normally associated with NK cells, such as NK1.1. Examples of NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8 cells. The TCR of NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)." Gamma delta T cells refer to a special population of a small subset of T cells that have different TCRs on their surface, unlike most T cells whose TCR consists of two glycoprotein chains denoted as α and β-TCR chains. The TCR in γδ T cells consists of a γ chain and a δ chain. γδ T cells have been found to play a role in immune surveillance and immunomodulation, to be an important source of IL-17, and to induce active CD8+ cytotoxic T cell responses. The term also includes "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Treg cells are typically Foxp3-positive CD4+ T cells, and can also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0067] The terms “natural killer cells” and “NK cells” are interchangeable and are used synonymously herein. As used herein, NK cells refer to differentiated lymphocytes having the CD16+CD56+ and / or CD57+TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express “auto” MHC / HLA antigens through activation of specific cytolytic enzymes, their ability to kill tumor cells or other disease cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0068] As used herein, the term “antigen” refers to any agent molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) to which a T cell receptor can bind. Antigens can also evoke an immune response. Examples of immune responses, though not limited to, may include antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It is readily apparent that antigens may be synthesized, derived from biological samples, or be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or fluids containing lysates.

[0069] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding domains may be synthetic polypeptides, enzymatically available polypeptides, or genetically engineered polypeptides, and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelid VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDR of an antibody, such as the FR3-CDR3-FR4 moiety, HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to antigens, and multispecific proteins including antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked to each other via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain, such as a single-chain Fv(scFv) or diabody, when the VH and VL domains are expressed as separate single-chains. Antigen-binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, for genetic engineering of bispecific and multispecific proteins.

[0070] The term “antibody” refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv(scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), intrabodies, minibodies, diabodies, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), and any of the epitope-binding fragments described above. The terms “antibody” and “antibody” also refer to covalent diabodies, such as those described in U.S. Patent Application Publication 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910. Antibodies useful as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. The immunoglobulin molecule may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or a subclass.

[0071] "Human antibody" means an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody includes heavy-chain and light-chain variable regions "derived" from a human sequence. Such exemplary systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. "Human antibody" typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the human antibody and the system used to obtain the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain, for example, a consensus framework sequence obtained from human framework sequence analysis described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0072] "Humanized antibody" means an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Since the framework of a humanized antibody can contain substitutions, the framework may not be an exact copy of the expressed human immunoglobulin or the human immunoglobulin germline gene sequence.

[0073] "Specifically binds", "specific binding", "specifically bound", or "binds" means that a proteinaceous molecule binds to an antigen or an epitope within the antigen with a higher affinity than to other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K D M or less, such as about 5 × 10 -8 M or less, about 1 × 10 -8 M or less, about 1 × 10 -9 M or less, about 1 × 10 -10 M or less, about 1 × 10 -11 M or less, or about 1 × 10 -12 M or less, and typically K D is at least 100-fold smaller than K D for binding to non-specific antigens (e.g., BSA, casein). In the context of the CD79b antigen described herein, "specific binding" means that a proteinaceous molecule binds to the CD79b antigen without detectably binding to the wild-type protein of which the CD79b antigen is a variant.

[0074] ​​The term "host cell" means any cell containing heterologous nucleic acids. Heterologous nucleic acids can be vectors (e.g., expression vectors). For example, a host cell may be a cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way for the production of substances by the cell, such as the expression of genes, DNA or RNA sequences, proteins, or enzymes. A suitable host can be determined. For example, a host cell may be selected based on the vector backbone and the desired outcome. As an example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate several types of vectors. Bacterial cells, SURE® competent cells, and SOLOPACK Gold cells, such as but not limited to DH5α, JM109, and KCB, can be used as host cells for vector replication and / or expression. Furthermore, bacterial cells such as E. coli LE392 can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.

[0075] The host cells of this disclosure include T cells and natural killer cells that contain a DNA or RNA sequence encoding a CAR and express the CAR on their cell surface. The host cells may be used to enhance T cell activity, natural killer cell activity, and for the treatment of cancer and autoimmune diseases.

[0076] The term "proliferation" refers to an increase in cell division, whether symmetric or asymmetric. The term "expansion" refers to the result of cell division and cell death.

[0077] The term "differentiation" refers to a method of reducing the capacity or proliferation of cells, or of transitioning cells to a more developmentally limited state.

[0078] The terms “expression” and “expression” mean enabling or causing the generation of information from a gene or DNA sequence, for example, producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed within or by a cell to form “expression products,” such as proteins. The expression product itself, for example, the resulting protein, can also be said to have been “expressed” by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0079] The term “transfection” means introducing “foreign” (i.e., exogenous or extracellular) nucleic acids into a cell using recombinant DNA technology. The term “genetic modification” means introducing “foreign” (i.e., exogenous or extracellular) genes, DNA, or RNA sequences into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence and may include regulatory or control sequences that are manipulably ligated to a polynucleotide encoding a chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by the cell’s genetic mechanisms. The gene or sequence may include non-functional sequences that do not have a known function. A host cell that receives and expresses introduced DNA or RNA is “genetically modified.” The DNA or RNA introduced into a host cell may be of any origin, including cells of the same genus or species as the host cell, or from a different genus or species.

[0080] The term "transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0081] The term “regulatory element” refers to any cis-acting genetic element that controls several aspects of the expression of a nucleic acid sequence. In some embodiments, the term “promoter” essentially includes the minimum sequence necessary to initiate transcription. In some embodiments, the term “promoter” includes the sequence for initiating transcription, plus additional sequences that can upregulate or downregulate transcription, which are commonly referred to as “enhancer elements” and “repressor elements,” respectively.

[0082] As used herein, the terms “operable linkage” or “operable linkage” and similar phrases, when used in relation to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequences that are functionally related to each other, respectively. For example, operational linkages of promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA). In some embodiments, operational linkages of nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, operational linkages of peptides refer to those in which functional domains are positioned at appropriate distances from each other to confer the intended function of each domain.

[0083] The terms “to treat” or “treatment” refer to therapeutic treatments aimed at delaying (reducing) unwanted physiological changes or diseases, or at producing beneficial or desired clinical outcomes during the treatment. Beneficial or desired clinical outcomes include, whether detectable or undetectable, relief of symptoms, reduction of disease severity, a stable (i.e., non-worsening) disease state, delay or slowing of disease progression, improvement or relief of disease status, and remission (whether partial or total). “Treatment” may also mean extending survival compared to the expected survival time if the subject were not treated. Subjects requiring treatment include those who already have unwanted physiological changes or diseases, as well as those who are prone to developing physiological changes or diseases.

[0084] As used herein, the term “subject / subject” refers to an animal. The terms “subject” and “patient” may be used interchangeably herein with respect to a subject. Thus, “subject” includes, as a patient, a human being treated for a disease or for the prevention of a disease. The methods described herein may be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Examples of mammals include, but are not limited to, rodents such as mice and hamsters, and lagomorphs such as rabbits. Mammals may be from the order Carnivora, which includes felines (cats) and canines (dogs). Mammals may be from the order Artiodactyla, which includes bovines (cats) and pigs (sea pigs), or from the order Perissodactyla, which includes equids (horses). Mammals may be from the order Primates, Ceboids, or Simoids (monkeys), or Haplorhini (humans and apes). In one embodiment, the mammal is a human.

[0085] "Enhancement," "promotion," "increase," "expansion," or "improvement" generally refer to the ability of the composition intended herein to produce, induce, or cause (i.e., downstream effect) a greater physiological response compared to the response caused by either the vehicle or the control molecule / composition. Measurable physiological responses may include, as will be evident from the understanding of the art and the description herein, an increase in T cell expansion, activation, effector function, persistence, and / or cancer cell killing ability. In certain embodiments, the "increased" or "enhanced" amount may be a "statistically significant" amount and may include increases of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (e.g., 500, 1000 times) (including all integers and decimals, such as 1.5, 1.6, 1.7, 1.8, etc.). The response is generated by the vehicle or the control composition.

[0086] "Decrease," "lower," "less," "reduce," or "weaken" generally refers to the ability of a composition intended herein to produce, induce, or cause a less physiological response (i.e., downstream effect) compared to a response caused by either the vehicle or the control molecule / composition. In certain embodiments, the amount "decreased" or "reduced" may be a "statistically significant" amount and may include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (intermediate and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) from a response caused by the vehicle, the control composition (reference response), or a response in a particular cell lineage.

[0087] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0088] "Recurrent" refers to the recurrence of the disease or its signs and symptoms after a period of improvement following prior treatment with medication.

[0089] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or it may become resistant during treatment.

[0090] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, ex vivo, or tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can be induced by infection with transforming viruses and the incorporation of new genomic nucleic acids, or by the uptake of exogenous nucleic acids, and may occur spontaneously or after exposure to carcinogens, thereby resulting in mutations of endogenous genes. Transformations / cancer are exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and ex vivo.

[0091] The term "effective" as applied to dosage or quantity refers to the amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject requiring it. When administering a combination of active ingredients, it should be noted that the effective dose of the combination may or may not include the amount of each individual ingredient that would be effective if administered individually. The exact required dose will vary depending on the subject's species, age, and overall health, the severity of the condition being treated, the specific drug used, and the mode of administration.

[0092] The terms “prevention,” “prevention,” or “prophylaxis” of a disease or disability mean preventing the disability from occurring in the subject.

[0093] In this specification, the terms “therapeutably effective dose” or “effective dose” as used interchangeably refer to the amount effective in obtaining the desired therapeutic outcome in the required dosage and duration. The therapeutically effective dose may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient’s health, reduction in tumor burden, cessation or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0094] As used herein in reference to the compositions, the term “pharmaceutically acceptable” means a molecular entity and other components of such a composition that are physiologically acceptable and do not typically produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means that it is approved for use in mammals, more specifically in humans, by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0095] As used herein, the term "protein" encompasses all types of modified proteins, including but not limited to natural and synthetic proteins, fusion proteins, and glycoproteins, including protein fragments of all lengths, as well as all other types of modified proteins (e.g., proteins obtained from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0096] The terms “nucleic acid,” “nucleotide,” and “polynucleotide” encompass both DNA and RNA unless otherwise specified. “Nucleic acid sequence” or “nucleotide sequence” means a nucleic acid sequence that codes for amino acids, and these terms may also refer to a nucleic acid sequence that includes any amino acid coding by a linker, or any amino acid coding portion added as a cloning artifact.

[0097] "Isolated" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides or polypeptides) that has been substantially separated and / or purified from other components of the system in which the molecules are produced, such as in recombinant cells, in addition to proteins that have undergone at least one purification or isolation step. "Isolated" refers to molecules that are substantially free from other cellular materials and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0098] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle to which a compound is administered together. Such pharmaceutical carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or aqueous solutions, physiological saline, and aqueous solutions of dextrose and glycerin are particularly preferred as carriers for injectable solutions. Alternatively, the carrier may be a solid dosage form carrier containing, but not limited to, one or more of the following: a binder (for compressed pills), a flow enhancer, a encapsulant, a flavoring agent, and a coloring agent. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0099] "Differentiation antigen group 79B protein" or "CD79b" refers to a known protein also known as B cell-specific glycoprotein B29, Ig-β, or AGM6. The B lymphocyte antigen receptor is a multimeric complex containing antigen-specific components and surface immunoglobulin (Ig), and it non-covalently associates with Ig-α and Ig-β. CD79b is the Ig-β protein of the B cell antigen component. All CD79b isoforms and variants are encompassed under "CD79b". The amino acid sequences of various isoforms can be obtained from GenBank accession numbers AAH32651.1, EAW94232.1, AAH02975.2, NP_000617.1, and NP_001035022.1. The amino acid sequence of the full-length CD79b sequence is shown below. The sequence includes an extracellular domain (residues 29-159) and a cytoplasmic domain (residues 181-229). MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (Sequence ID 282)

[0100] Chimeric antigen receptor This invention generally relates to the use of genetically modified T cells that stably express a desired chimeric antigen receptor. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide (scFv) containing an antigen-binding domain of an antibody linked to a T cell signaling domain. Characteristics of CARs include the ability to redirect T cell specificity and reactivity toward non-MHC restricted targets and to utilize the antigen-binding properties of monoclonal antibodies. Due to non-MHC restricted antigen recognition, T cells expressing CARs can recognize antigens independently of antigen processing, thus bypassing the main mechanism of tumor escape. Furthermore, when expressed in T cells, CARs do not favorably dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0101] The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as the "cytoplasmic signaling domain") which includes a functional signaling domain derived from a stimulating molecule as defined below. T cells expressing CARs are referred herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cells can be genetically modified to stably express an antibody-binding domain on their surface, conferring novel MHC-independent antigen specificity.

[0102] In some cases, T cells are genetically modified to stably express a CAR that combines the antigen-recognition domain of a specific antibody with the intracellular domain of a CD3-ζ chain or an FcγRI protein to form a single chimeric protein. In one embodiment, the stimulating molecule is a ζ chain associated with the T cell receptor complex.

[0103] The term "intracellular signaling domain" refers to the intracellular portion of a molecule as used herein. This is a functional part of a protein that acts by transmitting information within the cell to regulate cellular activity via signaling pathways defined by the generation of second messengers, or by acting as an effector in response to such messengers. Intracellular signaling domains generate signals that promote the immune effector function of CAR-containing cells (e.g., CAR-T cells). Examples of immune effector function in CAR-T cells include cytolytic activity and helper activity (including cytokine secretion).

[0104] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. An example of a primary intracellular signaling domain is one derived from a molecule involved in primary stimulation or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. An example of a co-stimulatory intracellular signaling domain is one derived from a molecule involved in co-stimulatory signaling or antigen-independent stimulation. For example, in the case of CAR-T cells, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0105] The primary intracellular signaling domain may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[0106] The terms "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" are defined as the protein provided as GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rabbits, primates, rodents, monkeys, and apes, and the "ζ stimulating domain" or alternatively "CD3-ζ stimulating domain" or "TCR-ζ stimulating domain" are defined as amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signals necessary for T cell activation. In one embodiment, the cytoplasmic domain of ζ includes residues 52-164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, and apes that are functional orthologues thereof. In one embodiment, the "ζ-stimulating domain" or "CD3-ζ-stimulating domain" is a sequence that has at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with the sequence provided as SEQ ID NO: 28 or SEQ ID NO: 41.

[0107] The term "costimulatory molecule" refers to an allobinding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells (e.g., proliferation, but not limited to T cell proliferation). Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0108] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83.

[0109] An intracellular signaling domain may include the entire intracellular portion of the molecule from which it originates, the entire intrinsic intracellular signaling domain, or functional fragments thereof.

[0110] The term "4-1BB" or alternatively "CD137" refers to the amino acid sequence provided as GenBank accession number AAA62478.2, or a member of the TNFR superfamily having equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. The "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. In one embodiment, the "4-1BB co-stimulatory domain" or the "CD137 co-stimulatory domain" is the sequence provided as SEQ ID NO: 27, or an equivalent residue from a non-human species, such as a mouse, rodent, monkey, or ape, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 40.

[0111] In one embodiment, a transmembrane domain naturally associated with one of the domains within the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex. In one example embodiment, the transmembrane domain includes a CD8α hinge domain.

[0112] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined herein. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-ζ, and / or CD28. CD28 is an important T cell marker in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and functions as a costimulatory immune checkpoint molecule. 4-1BB transmits potent costimulatory signals to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3-ζ associates with the TCR to generate signals and contains immune receptor tyrosine-based activation motifs (ITAMs). In another embodiment, the costimulatory molecule is MyD88 or CD40.

[0113] In one embodiment, the CAR comprises an intracellular hinge domain containing CD8 and an intracellular T cell receptor signaling domain containing CD28, 4-1BB, and CD3-ζ. In another embodiment, the CAR comprises an intracellular hinge domain and an intracellular T cell receptor signaling domain containing CD28, 4-1BB, and CD3-ζ, wherein the hinge domain comprises all or part of the extracellular region of CD8, CD4, or CD28, all or part of the antibody constant region, the FcRIIIA receptor, IgG hinge, IgM hinge, IgA hinge, IgD hinge, IgE hinge, or all or part of the Ig hinge. The IgG hinge may be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0114] The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as the "cytoplasmic signaling domain") which includes a functional signaling domain derived from, for example, a stimulating molecule as defined below.

[0115] In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulating molecule. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulating molecule.

[0116] The CARs of the present invention can be designed to contain the CD28 and / or 4-1BB signaling domain by themselves, or to be combined with any other desired cytoplasmic domain useful in the context of the CARs of the present invention. In one embodiment, the cytoplasmic domain of the CAR may further contain the CD3-ζ signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, the CD3-ζ, 4-1BB, and CD28 signaling modules, as well as combinations thereof. Accordingly, the present invention provides CAR T cells and methods of using them for adoptive therapy.

[0117] This disclosure further provides variants (e.g., functional variants) of CARs, nucleic acids, polypeptides, and proteins described herein. A “variant” means a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitution, insertion, or deletion. As used herein, the term “functional variant” means a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, polypeptide, or protein, and these functional variants retain the biological activity of the variant CAR, polypeptide, or protein. Functional variants include, for example, variants of CARs, polypeptides, or proteins (parent CARs, polypeptides, or proteins) described herein, and retain the ability to recognize target cells to the same degree, the same degree, or a higher degree than the parent CAR, polypeptide, or protein. With respect to the parent CAR, polypeptide, or protein, the functional variant may have an amino acid sequence that is identical to, for example, the parent CAR, polypeptide, or protein by at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0118] A functional variant may include, for example, an amino acid sequence of a parent CAR, polypeptide, or protein having at least one conserved amino acid substitution. In another embodiment, a functional variant may include an amino acid sequence of a parent CAR, polypeptide, or protein having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution cannot suppress or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the parent CAR, polypeptide, or protein.

[0119] The amino acid substitutions of CARs in the present invention may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions may include acidic amino acids substituted with another acidic amino acid (e.g., Asp or Glu), amino acids with nonpolar side chains substituted with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids substituted with another basic amino acid (e.g., Lys, Arg), and amino acids with polar side chains substituted with another amino acid having a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0120] CARs, polypeptides, or proteins may essentially consist of a specific amino acid sequence or a sequence described herein, and as a result, other components, such as other amino acids, do not substantially alter the biological activity of the functional variant.

[0121] The CARs, polypeptides, and proteins (including functional portions and functional variants) of embodiments of this disclosure may be of any length, i.e., may contain any number of amino acids, provided that the CARs, polypeptides, or proteins (or functional portions or their functional variants) retain their biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells (e.g., cancer cells) in a host, or the ability to treat or prevent disease in a host. For example, polypeptides may have amino acid lengths of about 50 to about 5000, for example, about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000 or more. The polypeptides of the present invention also include oligopeptides.

[0122] The CARs, polypeptides, and proteins (including the functional portions and functional variants of the present invention) of embodiments of the present invention may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β- Examples include hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, and α-tert-butylglycine.

[0123] The CARs, polypeptides, and proteins (including functional moieties and functional variants) of embodiments of the present invention can undergo post-translational modifications. These can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via disulfide crosslinks, for example, or converted to acid addition salts. In some embodiments, these are dimerized, polymerized, or conjugated.

[0124] The CARs, polypeptides, and / or proteins (including functional portions and functional variants thereof) of embodiments of the present invention can be obtained by methods known in the art. Preferred methods for the de novo synthesis of polypeptides and proteins are described in reference to Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Arcel Dekker, Inc., 2000; and Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Polypeptides and proteins can also be produced by recombination using the nucleic acids described herein using standard recombination methods. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some of the CARs, polypeptides, and proteins (including functional portions and their functional variants) of the present invention can be isolated and / or purified from sources such as plants, bacteria, insects, and mammals. Methods for isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and / or proteins (including functional portions and their functional variants) described herein can be commercially synthesized. In this regard, CARs, polypeptides, and proteins can be synthesized, recombinant, isolated, and / or purified.

[0125] Examples of modified nucleotides that can be used to produce recombinant nucleic acids used to produce the polypeptides described herein include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N 6 - Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquosin, 5"-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyl adenine, uracil-5-oxyacetic acid (v), weybutoxosin, pseudouracil, quosin, beta-D-galactosylquosin, inosine, N 6 Examples include, but are not limited to, isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0126] Nucleic acids may include any isolated or purified nucleotide sequence encoding a CAR, polypeptide, or protein, or a functional portion or a functional variant thereof. Alternatively, a nucleotide sequence may include a nucleotide sequence degenerated into any of the sequences, or a combination of degenerate sequences.

[0127] Some embodiments of the present invention also provide isolated or purified nucleic acids comprising a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes under stringent conditions to any of the nucleotide sequences of the nucleic acids described herein.

[0128] Nucleotide sequences that hybridize under stringent conditions may hybridize under high-stringency conditions. “High-stringency conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectable amount stronger than non-specific hybridization. High-stringency conditions include conditions that distinguish polynucleotides having precisely complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that end up having several sub-regions (e.g., 3–12 bases) that match the nucleotide sequence. Such complementary sub-regions melt more readily than full-length complements of 14–17 or more bases, and high-stringency hybridization makes them readily identifiable. Relatively high-stringency conditions include low-salt and / or high-temperature conditions, such as those provided by approximately 0.02–0.1 M NaCl or equivalent at a temperature of approximately 50–70°C. Such highly stringent conditions tolerate little to no mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the CARs described herein. It is generally understood that the conditions can be made more stringent by the addition of gradually increasing amounts of formamide.

[0129] In one embodiment, the nucleic acids of the present invention can be incorporated into a recombinant expression vector. This disclosure provides recombinant expression vectors comprising any of the nucleic acids of the present invention. As used herein, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide, or peptide by a host cell, wherein the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is brought into contact with the cell under conditions sufficient to express mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein as a whole do not exist in nature. However, parts of the vectors may exist in nature. The recombinant expression vectors described may contain any type of nucleotide, including but not limited to DNA and RNA (which may be single-stranded or double-stranded, synthesized, or partially obtained from natural sources, and may contain natural, unnatural, or modified nucleotides). Recombinant expression vectors may contain naturally occurring nucleotide linkages, or non-natural nucleotide linkages, or both types of linkages. Nucleotides or internucleotide links that do not exist naturally or have been modified do not inhibit the transcription or replication of the vector.

[0130] In one embodiment, the recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for the reproduction and proliferation of plasmids and viruses, or for expression, or both. Vectors may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) may be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors, such as gamma retroviral vectors.

[0131] In one embodiment, the recombinant expression vector of the present invention is prepared, for example, using standard recombinant DNA techniques described in Sambrook et al. (above) and Ausubel et al. (above). The circular or linear expression vector construct may be prepared to include a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system may be derived, for example, from ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0132] Recombinant expression vectors may, as appropriate, include regulatory sequences such as transcription and translation start and stop codons that are specific to the type of host into which the vector is introduced (e.g., bacteria, plants, fungi, or animals), taking into consideration whether the vector is DNA-based or RNA-based.

[0133] Recombinant expression vectors may contain one or more marker genes that enable selection of transformed or transfected hosts. Marker genes may include those for biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.) or for supplementation to provide protonutrient in nutritionally dependent hosts. Suitable marker genes for the described expression vectors include, for example, neomycin / G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0134] Recombinant expression vectors may include a native or conventional promoter operably ligated to a nucleotide sequence encoding a CAR, polypeptide, or protein (including functional portions and functional variants thereof), or to a nucleotide sequence complementary to or hybridizing with a CAR, polypeptide, or protein-encoding nucleotide sequence. The choice of promoter (e.g., strong, weak, tissue-specific, inducible, and developmentally specific) is within the scope of the art. Similarly, combining nucleotide sequences with promoters is also within the scope of the art. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, RSV promoters, SV40 promoters, or promoters found in long terminal repeats of murine stem cell viruses.

[0135] Recombinant expression vectors may include one or more additional regulatory elements, such as enhancer elements, 5' and 3' UTRs, or terminator sequences, which are operably ligated to a nucleotide sequence encoding a CAR, polypeptide, or protein (including functional portions and functional variants thereof), or to a nucleotide sequence that is complementary to or hybridizes with a nucleotide sequence encoding a CAR, polypeptide, or protein.

[0136] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0137] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to a drug (e.g., a pharmacokinetic drug) to cells expressing the gene, causing the cells to die when they come into contact with or are exposed to the drug. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0138] Conjugates comprising any of the following—CARs, polypeptides, or proteins (including either their functional portions or variants), host cells, nucleic acids, recombinant expression vectors, host cell populations, or antibodies, or their antigen-binding portions, such as bioconjugates—are within the scope of the present invention. Conjugates and methods for synthesizing them are generally known in the art (see, for example, Hudecz, F., Methods Mol. Biol. 298:209-223 (2005) and Kirin et al., Inorg Chem. 44(15):5405-5415 (2005)).

[0139] One embodiment of the present invention further provides an antibody or antigen-binding moiety that binds, for example, specifically to the epitope of the CAR of the present invention.

[0140] Antibodies can be any type of immunoglobulin known in the art. Immunoglobulins can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of vertebrate species can be assigned to one of two types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Antibodies can be of any class or isotype.

[0141] Antibodies include monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies; polyclonal antibodies; antigen-binding fragments; bispecific or multispecific antibodies; monomeric, dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; domain antibodies; and immunoglobulin molecules in any other modified configuration containing antigen-binding sites of the required specificity. Antibodies may be naturally occurring antibodies, such as those isolated and / or purified from mammals (e.g., mice, primates, rabbits, goats, horses, chickens, hamsters, humans, etc.). Alternatively, antibodies may be genetically engineered antibodies.

[0142] Humanized antibodies have antigen-binding sites derived from non-human species, and their variable region framework is derived from human immunoglobulin sequences. Human antibodies have heavy and light chain variable regions in which both the framework and antigen-binding sites are derived from human sequences.

[0143] Furthermore, antibodies may have any level of affinity or binding activity to the functional portion of CAR. In some embodiments, antibodies have an affinity (K) within a certain range. D) can bind to the CD79b antigen. In one embodiment of the present invention, and in each of the numbered embodiments listed below and in some of the embodiments of all embodiments, the antibody has high affinity, for example, about 10, as determined by surface plasmon resonance or binding equilibrium exclusion (Kinexa) as performed by those skilled in the art. -7 For KD values ​​less than or equal to M, for example, without limitation, 1 to 9.9 (or any range or value that falls within this range, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 It binds to the CD79b antigen with a KD of M, or any range or value within this range. An example of affinity is 1 × 10⁻¹⁰. -8 It is less than or equal to M. Another example of affinity is 1 × 10 -9 It is M or less.

[0144] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assay.

[0145] Suitable methods for producing antibodies are known in the art. For example, the standard hybridoma method is described, for instance, in Kohler and Milstein, Eur. J. Immunol., 5, 511519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 36167 (1984), and Roder et al., Methods Enzymol., 121, 14067 (1986)), and bacteriophage vector expression systems (see, for example, Huse et al., Science, 246, 1275-81 (1989)). Furthermore, methods for producing antibodies in non-human animals are described, for example, in U.S. Patents No. 5,545,806, No. 5,569,825, and No. 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266(A1).

[0146] Antibodies can also be generated using phage display. In this regard, phage libraries encoding the antigen-binding variable (V) domain of an antibody can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al. (above) and Ausubel et al. (above)). Phages encoding variable regions with the desired specificity are selected for specific binding to the desired antigen (i.e., CD79b), and a complete or partial antibody containing the selected variable domain is reconstituted. The nucleic acid sequence encoding the reconstituted antibody is introduced into a suitable cell line, such as myeloma cells used for hybridoma production, resulting in the secretion of an antibody with monoclonal antibody characteristics by the cells (see, e.g., Janeway et al. (above), Huse et al. (above), and U.S. Patent No. 6,265,150).

[0147] Antibodies can be produced by transgenic mice that are transgenic of specific heavy and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Patents No. 5,545,806 and No. 5,569,825, and by Janeway et al., cited above.

[0148] Methods for generating humanized antibodies are known in the art and are described, for example, by Janeway et al., cited above, U.S. Patents 5,225,539, 5,585,089, and 5,693,761, European Patent No. 0239400(B1), and British Patent No. 2188638. Humanized antibodies can also be generated using the antibody resurfacing technique described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

[0149] When used herein, antibodies may be multiple or single chains, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0150] In some embodiments, the antibody is a bispecific antibody. The VL and / or VH regions of an existing antibody, or newly identified VL and VH regions as described herein, may be genetically engineered to produce a bispecific full-length antibody. Such bispecific antibodies can be produced by modifying CH3 interactions in antibody Fc to form bispecific antibodies using techniques such as those described below: U.S. Patent No. 7,695,936, International Publication No. 04 / 111233, U.S. Patent Publication No. 2010 / 0015133, U.S. Patent Publication No. 2007 / 0287170, International Publication No. 2008 / 119353, U.S. Patent Publication No. 2009 / 0182127, U.S. Patent Publication No. 2010 / 0286374, U.S. Patent Publication No. 2011 / 0123532, International Publication No. 2011 / 131746, International Publication No. 2011 / 143545, or U.S. Patent Publication No. 2012 / 0149876. For example, the bispecific antibody of the present invention may be produced by introducing asymmetric mutations into the CH3 region of two monospecific homodimer antibodies in a cell-free environment, according to the method described in International Publication No. 2011 / 131746, and forming a bispecific heterodimer antibody from two parent monospecific homodimer antibodies under reducing conditions that isomerize the disulfide bond. In this method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are genetically engineered to have a specific substitution in the CH3 domain that promotes heterodimer stability, but these antibodies are incubated together under reducing conditions sufficient to isomerize the disulfide bond at the cysteine ​​in the hinge region, thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine.For example, incubation for at least 90 minutes may be used at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, for example, pH 7.0 or pH 7.4.

[0151] The term "antibody fragment" refers to at least one portion of an intact antibody or its recombinant variant that retains the antigen-binding properties of the parent full-length antibody. This refers to, for example, an antigen-binding domain (e.g., the antigen-determining variable region of an intact antibody) that is sufficient to confer recognition and binding (e.g., specific binding of the antibody fragment to a target such as an antigen). "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies (scFv), linear antibodies, single-domain antibodies (either VL or VH) such as sdAb, camel VHH domains, and multispecific antibodies formed from antibody fragments.

[0152] The term "scFv" refers to a protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region. In some embodiments, the light chain variable region and the heavy chain variable region are sequentially linked via a short, flexible polypeptide linker, allowing the scFv to be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it originated. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in any order, and for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL-linker-VH or a VH-linker-VL.

[0153] The VH and VL domains identified herein can be incorporated into the scFv format, and the binding and thermal stability of the resulting scFv to CD79b can be evaluated using known methods. Binding can be evaluated using ProteOn XPR36, Biacore3000, or KinExA instruments, ELISA, or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFv or lysed cells containing E. coli supernatant or expressed scFv. Measured affinity of test scFV to CD79b may differ when measured under different conditions (e.g., molar osmotic pressure, pH). Therefore, affinity and other binding parameters (e.g., K) may differ. D , K on , K off The measurement of thermal stability is usually performed using standard conditions and standardized buffer. Thermal stability can be evaluated by heating the test scFv at a high temperature such as 50°C, 55°C, or 60°C for a period of time such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, and measuring the binding of the test scFv to CD79b. An scFv that retains equivalent binding to CD79b when compared to an unheated scFv sample is said to be thermally stable.

[0154] The cD79b antigen-binding domain may contain sequences encoding variants that exhibit improved thermal stability compared to the parent antibody SN8. Genetically engineered sites that can confer improved thermal stability include residues M12, I20, R40, and A79 in VH (residue numbering according to SN8_VH in SEQ ID NO: 283), and L4, D32, F51, V82, and A87 in VL (residue numbering according to SN8_VL in SEQ ID NO: 284).

[0155] In recombinant expression systems, the linker is a peptide linker and may contain any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker needs to be of an appropriate length to link VH and VL in such a way that they form precise higher-order structures relative to each other, in order to maintain desired activity, such as binding to CD79b.

[0156] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker is approximately 10 to 40 amino acids long. In some embodiments, the linker is approximately 10 to 35 amino acids long. In some embodiments, the linker is approximately 10 to 30 amino acids long. In some embodiments, the linker is approximately 10 to 25 amino acids long. In some embodiments, the linker is approximately 10 to 20 amino acids long. In some embodiments, the linker is approximately 15 to 20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acid lengths. In some embodiments, the linker is 16 amino acid lengths. In some embodiments, the linker is 17 amino acid lengths. In some embodiments, the linker is 18 amino acid lengths. In some embodiments, the linker is 19 amino acid lengths. In some embodiments, the linker is 20 amino acid lengths. In some embodiments, the linker is 21 amino acid lengths. In some embodiments, the linker is 22 amino acid lengths. In some embodiments, the linker is 23 amino acid lengths. In some embodiments, the linker is 24 amino acid lengths. In some embodiments, the linker is 25 amino acid lengths. In some embodiments, the linker is 26 amino acid lengths. In some embodiments, the linker is 27 amino acid lengths. In some embodiments, the linker is 28 amino acid lengths. In some embodiments, the linker is 29 amino acid lengths.In some embodiments, the linker is 30 amino acid lengths. In some embodiments, the linker is 31 amino acid lengths. In some embodiments, the linker is 32 amino acid lengths. In some embodiments, the linker is 33 amino acid lengths. In some embodiments, the linker is 34 amino acid lengths. In some embodiments, the linker is 35 amino acid lengths. In some embodiments, the linker is 36 amino acid lengths. In some embodiments, the linker is 37 amino acid lengths. In some embodiments, the linker is 38 amino acid lengths. In some embodiments, the linker is 39 amino acid lengths. In some embodiments, the linker is 40 amino acid lengths. Exemplary linkers that can be used are glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and other flexible linkers.

[0157] Other linker sequences may include immunoglobulin hinge regions, CL, or CH1 portions derived from immunoglobulin heavy or light chain isotypes. Exemplary linkers that may be used are shown in Table 1. Additional linkers are described, for example, in International Publication No. 2019 / 060695.

[0158] In some embodiments, scFv includes a VH, a linker, and a VL (VH-linker-VL) from the N-terminus to the C-terminus.

[0159] In some embodiments, scFv includes a VL, a linker, and a VH (VL-linker-VH) from the N-terminus to the C-terminus.

[0160] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 42.

[0161] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 43.

[0162] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 44.

[0163] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 45.

[0164] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 46.

[0165] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 47.

[0166] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 48.

[0167] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 49.

[0168] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 50.

[0169] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 51.

[0170] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 52.

[0171] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 53.

[0172] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 54.

[0173] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 55.

[0174] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 56.

[0175] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 57.

[0176] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 58.

[0177] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 59.

[0178] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 60.

[0179] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 61.

[0180] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 62.

[0181] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 63.

[0182] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 64.

[0183] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 65.

[0184] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 66.

[0185] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 67.

[0186] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 68.

[0187] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 69.

[0188] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 70.

[0189] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 71.

[0190] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 72.

[0191] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 73.

[0192] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 74.

[0193] [Table 1]

[0194] One embodiment of the present invention also provides an antigen-binding moiety of any of the antibodies described herein. The antigen-binding moiety may be any moiety having at least one antigen-binding site, such as Fab, F(ab')2, dsFv, sFv, diabody, and triabody.

[0195] In some embodiments, the antigen-binding fragment is a domain antibody (dAb) comprising (e.g., consisting of) heavy chain complementarity determining regions (HCDR) 1, 2, and / or 3, light chain complementarity determining regions (LCDR) 1, 2, and / or 3, a heavy chain variable region (VH), or a light chain variable region (VL), Fab, F(ab')2, Fd, and Fv fragments, and either one VH domain or one VL domain. The VH and VL domains may be linked together via a linker, such as a synthetic linker.

[0196] Complementarity determining regions (CDRs) are antigen-binding sites in antibodies. CDRs can be defined using various terms: (i) Three complementarity determining regions (CDRs) in the VH (Hyperhelium) (HCDR1, HCDR2, HCDR3) and three in the VL (Vitalis Limb) (LCDR1, LCDR2, LCDR3) are based on sequence diversity (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The "hypervariable regions," "HVR," or "HV," consisting of three (H1, H2, H3) within the VH and three (L1, L2, L3) within the VL, refer to regions of antibody variable domains that are structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The International ImMunoGeneTics (IMGT) database (http: / / imgt_org) provides standard numbering and definitions of antigen-binding sites. The correspondence between CDR, HV, and IMGT summaries is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. As used herein, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” include CDRs as defined by any of the methods described above by Kabat, Chothia, or IMGT, unless otherwise specified herein.

[0197] Furthermore, antibodies or their antigen-binding moieties may be modified to include detectable labels such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0198] Examples of detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron density reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescent quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatases, peroxidases, luciferases, electron donors / receptors, acridinium esters, and colorimetric substrates.

[0199] Detectable labels may spontaneously emit a signal, for example, when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal as a result of stimulation by an external field. Suitable dyes include any commercially available dyes such as 5(6)-carboxyfluorescein, IRDye 680RD maleimide, or IRDye 800CW, or ruthenium polypyridyl dyes. Suitable fluorophores include fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594, Alexa647), near-infrared (NIR) (700-900 nm) fluorescent dyes, as well as carbocyanin and aminostyryl dyes.

[0200] Nucleic acids comprising nucleotide sequences encoding any of the CARs, polypeptides, or proteins (including functional portions and functional variants thereof) described herein are provided by the present disclosure.

[0201] A portion of a CAR that includes an antibody or an antibody fragment thereof may exist in various forms, in which case the antigen-binding domain is expressed as part of contiguous polypeptide chains such as, for example, single-domain antibody fragments (sdAbs), scFvs, and human chimeric or humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y., Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423426). In one aspect, the antigen-binding domain of the CAR composition of the present invention includes an antibody fragment. In one aspect, the CAR includes an antibody fragment that includes an scFv.

[0202] The term "recombinant antibody" refers to an antibody generated using recombinant DNA techniques, such as an antibody expressed by a bacteriophage or yeast expression system. The term should be interpreted to mean an antibody that has been generated by synthesis of a DNA molecule encoding the antibody, where this DNA molecule expresses an antibody protein or an amino acid sequence that specifies an antibody, and in which case the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence techniques that are available and known in the art.

[0203] The term “antigen” refers to a molecule that triggers an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA, by containing a nucleotide sequence or partial nucleotide sequence that codes for a protein that triggers an immune response, codes for an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not have to be coded solely by the full-length nucleotide sequence of a gene. It is clear that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences, arranged in various combinations, code for polypeptides that trigger a desired immune response.

[0204] In one embodiment, the disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain binds to the CD79b antigen.

[0205] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is In one embodiment, the extracellular antigen-binding domain is a) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 1, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 19, b) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 1, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 20, c) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 2, and light chain complementarity determining regions (CDR)2, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 19 d) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 2, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 20, e) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 3, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 20, f) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 4, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 19, g) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 5, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 22, h) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 5, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 23, i) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 6, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 24, j) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 7, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 26, k) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 8, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 25, l) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 9, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 27, m) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 10, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 28, n) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 11, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 29 o) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 12, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 30, p) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 13, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 31, q) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 14, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 32, r) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 15, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 33, s) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 16, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 34, t) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 16, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 35, u) Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 17, and light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 33, or v) Including the heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 18, and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 36.

[0206] In one embodiment, the extracellular antigen-binding domain is This includes the heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of SEQ ID NO: 14, and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of SEQ ID NO: 32.

[0207] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The heavy chain CDR1 comprises amino acid sequences selected from the group consisting of SEQ ID NOs. 208, 216, 222, 228, 232, 238, 242, 248, 253, 257, 263, 268, and 274, as well as their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0208] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The heavy chain CDR2 comprises amino acid sequences selected from the group consisting of SEQ ID NOs. 209, 217, 223, 233, 239, 243, 249, 254, 258, 269, and 275, as well as their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0209] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The heavy chain CDR3 comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 210, 218, 224, 229, 234, 240, 244, 250, 255, 259, 264, 270, and 276, as well as their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0210] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is Heavy chain complementarity determination region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 208, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 209, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 210, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 216, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 217, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 218. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 228, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 217, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 229, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 232, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 233, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 234. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 238, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 239, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 240, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 242, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 244, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 248, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 249, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 250, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 253, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 254, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 255. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 257, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 258, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 259. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 263, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 264. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 268, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 269, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 270, or It comprises heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 274, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 275, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 276. The extracellular antigen-binding domain binds to the CD79b antigen.

[0211] In one embodiment, the CAR of this disclosure comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 257, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 258, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 259.

[0212] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The light chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 211, 214, 215, 219, 225, 230, 235, 241, 245, 251, 260, 265, 271, and 277, as well as their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0213] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The light chain CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 212, 220, 226, 231, 236, 246, 261, 266, and 272, as well as their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0214] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is The light chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 221, 227, 237, 247, 252, 256, 262, 267, 273, or 278, and their conserved modifications, and the extracellular antigen-binding domain binds to the CD79b antigen.

[0215] In one embodiment, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, light chain CDR2 having the amino acid sequence of SEQ ID NO: 212, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, light chain CDR2 having the amino acid sequence of SEQ ID NO: 220, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 230, light chain CDR2 having the amino acid sequence of SEQ ID NO: 231, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 221. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 235, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 245, light chain CDR2 having the amino acid sequence of SEQ ID NO: 246, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 252. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, light chain CDR2 having the amino acid sequence of SEQ ID NO: 236, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 256. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 262. Light chain CDR1 having the amino acid sequence of SEQ ID NO: 265, light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, light chain CDR2 having the amino acid sequence of SEQ ID NO: 272, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 273, or It comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 266, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 278. The extracellular antigen-binding domain binds to the CD79b antigen.

[0216] In one embodiment, the CAR of the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is It includes a light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 262.

[0217] In one embodiment, the extracellular antigen-binding domain is a) Sequence numbers 208, 209, 210, 211, 212, and 213 respectively, b) Sequence numbers 208, 209, 210, 214, 212, and 213 respectively, c) Sequence numbers 208, 209, 210, 215, 212, and 213 respectively, d) Sequence numbers 216, 217, 218, 219, 220, and 221, respectively e) Sequence numbers 222, 223, 224, 225, 226, and 227, respectively f) Sequence numbers 228, 217, 229, 230, 231, and 221 respectively, g) Sequence numbers 232, 233, 234, 235, 236, and 237, respectively h) Sequence numbers 238, 239, 240, 241, 226, and 227 respectively, i) Sequence numbers 242, 243, 244, 245, 246, and 247, respectively j) Sequence numbers 248, 249, 250, 251, 236, and 252, respectively k) Sequence numbers 253, 254, 255, 251, 236, and 256 respectively, l) Sequence numbers 257, 258, 259, 260, 261, and 262, respectively m) Sequence numbers 263, 243, 264, 265, 266, and 267 respectively, n) Sequence numbers 268, 269, 270, 271, 272, and 273, respectively, or o) comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, each having the amino acid sequences of SEQ ID NOs. 274, 275, 276, 277, 266, and 278, respectively.

[0218] In one embodiment, the extracellular antigen-binding domain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, each having the amino acid sequences of SEQ ID NOs. 257, 258, 259, 260, 261, and 262, respectively.

[0219] In one embodiment, the extracellular antigen-binding domain is This includes a heavy chain variable domain (HCVH) containing an amino acid sequence selected from SEQ ID NOs: 1 to 18, or a light chain variable domain (LCVL) containing an amino acid sequence selected from SEQ ID NOs: 19 to 36, or a combination of an HCVH containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18 and an LCVL containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 36.

[0220] In one embodiment, the extracellular antigen-binding domain is A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 4, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 23, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 25, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 27, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 28, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 29, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 33, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 34, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 35, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 33, or It includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 36.

[0221] In one embodiment, the extracellular antigen-binding domain is It includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 32.

[0222] In one embodiment, the extracellular antigen-binding domain is A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 1, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 19, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 1, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 20, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 2, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 19, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 20, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 20, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 3, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 20, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 4, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 19, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 5, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 22, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 5, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 23, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 6, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 24, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 7, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 26, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 8, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 25, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 9, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 27, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 10, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 28, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 11, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 29, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 12, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 30, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 13, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 31, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 14, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 32, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 15, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 33, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 16, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 34, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 16, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 35, A heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 17, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 33, or The device includes a heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 18, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 36.

[0223] In one embodiment, the extracellular antigen-binding domain is The device includes a heavy chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 14, and a light chain variable region containing an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 32.

[0224] In one embodiment, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide between the light chain variable region and the heavy chain variable region. In certain embodiments, the extracellular antigen-binding domain is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 75 to 118, which specifically binds to a CD79b polypeptide (e.g., a human CD79b polypeptide having the amino acid sequence described herein, or a fragment thereof). In some embodiments, the linker polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 42 to 74.

[0225] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In one embodiment, the linker polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 42.

[0226] In one embodiment, scFv includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 75 to 118. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 75. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 76. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 77. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 78. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 79. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 80. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 81. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 82. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 83. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 84. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 85. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 86. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO. 87. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 88. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 89. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 90. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 91. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 92. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 93. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 94. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 95. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 96. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 97. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 98. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 99. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 100. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 101. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 102. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 103. In another embodiment, scFv contains the amino acid sequence of SEQ ID NO: 104.In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 105. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 106. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 107. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 108. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 109. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 110. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 111. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 112. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 113. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 114. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 115. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 116. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 117. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 118.

[0227] In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with a sequence selected from the group consisting of sequence numbers 75 to 118. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 75. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 76. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 77. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 78. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 79.In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 80. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 81. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 82. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 83. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 84. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 85.In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 86. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 87. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 88. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 89. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 90. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 91.In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 92. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 93. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 94. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 95. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 96. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 97.In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 98. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 99. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 100. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 101. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 102. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 103.In one embodiment, scFv contains an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 104. In one embodiment, scFv contains an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 105. In one embodiment, scFv contains an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, with sequence number 106. The scFv includes an amino acid sequence having at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 107. In one embodiment, the scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 108. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 109. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 110. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 111. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 112.In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 113. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 114. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 115. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 116. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 117. In one embodiment, scFv includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with sequence number 118.

[0228] In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 113.

[0229] In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. In some embodiments, the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 37. In one embodiment, the signal polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 37.

[0230] In one embodiment, the disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-162. Another feature of a CAR having an extracellular antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-162 is that the extracellular antigen-binding domain binds to the CD79b antigen.

[0231] In one embodiment, the intracellular signaling domain includes polypeptide components selected from the group consisting of TNF receptor superfamily member 9 (CD137), T cell surface glycoprotein CD3ζ chain (CD3z), differentiation cluster (CD27), differentiation cluster superfamily member (e.g., CD28 or inducible T-cell co-stimulator, ICOS), and combinations thereof.

[0232] In one embodiment, component CD137 includes the amino acid sequence of SEQ ID NO: 40. In one embodiment, component CD137 includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 40.

[0233] In one embodiment, the CD3z component includes the amino acid sequence of SEQ ID NO: 41. In one embodiment, the CD3z component includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 41.

[0234] In one embodiment, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 163. In one embodiment, the intracellular signaling domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 163.

[0235] In one embodiment, the transmembrane domain comprises a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 39. In one embodiment, the CD8a-TM polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 39.

[0236] In one embodiment, the transmembrane domain includes at least (multiple) transmembrane regions of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. In another embodiment, the transmembrane domain includes at least the ζ, η, or FcεR1γ and β, MB1(Igα), B29, or CD3-γ, ζ, or η transmembrane domain. In yet another embodiment, the transmembrane domain is synthetic and includes mainly hydrophobic residues such as a triplet of leucine and valine, phenylalanine, or tryptophan.

[0237] In one embodiment, the CAR further includes a hinge region that links the transmembrane domain to an extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region includes the amino acid sequence of SEQ ID NO: 38. In some embodiments, the CD8a hinge region includes an amino acid sequence that has at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 38. In some embodiments, the hinge region includes the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 285) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with EPKSCDKTHTCPPCP (SEQ ID NO: 285). In some embodiments, the hinge region includes the sequence ERKCCVECPPCP (SEQ ID NO: 286) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with ERKCCVECPPCP (SEQ ID NO: 286). In some embodiments, the hinge region includes the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 287) or includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 287).In some embodiments, the hinge region includes the sequence ESKYGPPCPSCP (Sequence ID 288) or includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with ESKYGPPCPSCP (Sequence ID 288).

[0238] In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 119 to 162. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 119. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 120. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 121. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 122. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 123. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 124. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 125. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 126. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 127. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 128. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 129. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 130. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 131. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 132. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 133. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 134. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 135. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 136. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 137. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 138. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 139. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 140. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 141. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 142.In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 143. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 144. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 145. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 146. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 147. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 148. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 149. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 150. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 151. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 152. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 153. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 154. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 155. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 156. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 157. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 158. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 159. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 160. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 161. In one embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 162.

[0239] In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 119. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NOs. 119. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 120. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 121. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 122.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 123. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 124. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 125. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 126. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 127. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 128.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 129. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 130. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 131. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 132. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 133. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 134.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 135. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 136. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 137. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 138. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 139. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 140.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 141. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 142. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 143. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 144. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 145. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 146.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 147. In one embodiment, the extracellular antigen-binding domain comprises at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90 amino acids with SEQ ID NO: 148. The extracellular antigen-binding domain includes an amino acid sequence having at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 149. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 150. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 151. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 152. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 153.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 154. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 155. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 156. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 157. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 158. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 159.In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 160. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 161. In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 162.

[0240] In one embodiment, the extracellular antigen-binding domain includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 157.

[0241] CAR constructs and immune-responsive cells expressing CARs In one embodiment, the disclosure provides cells expressing a nucleic acid molecule encoding a CAR. In one embodiment, the CAR of the disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164 to 207. In some embodiments, the CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 164, or the amino acid sequence of SEQ ID NO: 164. In some embodiments, the CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 165, or the amino acid sequence of SEQ ID NO: 165. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 166, or the amino acid sequence of SEQ ID NO: 166. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 167, or the amino acid sequence of SEQ ID NO: 167.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 168, or the amino acid sequence of SEQ ID NO: 168. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 169, or the amino acid sequence of SEQ ID NO: 169. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 170, or the amino acid sequence of SEQ ID NO: 170. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 171, or the amino acid sequence of SEQ ID NO: 171. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 172, or the amino acid sequence of SEQ ID NO: 172.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 173, or the amino acid sequence of SEQ ID NO: 173. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 174, or the amino acid sequence of SEQ ID NO: 174. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 175, or the amino acid sequence of SEQ ID NO: 175. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 176, or the amino acid sequence of SEQ ID NO: 176. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 177, or the amino acid sequence of SEQ ID NO: 177.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 178, or the amino acid sequence of SEQ ID NO: 178. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 179, or the amino acid sequence of SEQ ID NO: 179. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 180, or the amino acid sequence of SEQ ID NO: 180. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 181, or the amino acid sequence of SEQ ID NO: 181. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 182, or the amino acid sequence of SEQ ID NO: 182.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 183, or the amino acid sequence of SEQ ID NO: 183. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 184, or the amino acid sequence of SEQ ID NO: 184. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 185, or the amino acid sequence of SEQ ID NO: 185. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 186, or the amino acid sequence of SEQ ID NO: 186. In some embodiments, the CAR of the present disclosure includes at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, with SEQ ID NO: 187. The CAR of the present disclosure includes an amino acid sequence having at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the amino acid sequence of SEQ ID NO: 187. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 188, or the amino acid sequence of SEQ ID NO: 188. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 189, or the amino acid sequence of SEQ ID NO: 189. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 190, or the amino acid sequence of SEQ ID NO: 190. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 191, or the amino acid sequence of SEQ ID NO: 191. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 192, or the amino acid sequence of SEQ ID NO: 192.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 193, or the amino acid sequence of SEQ ID NO: 193. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 194, or the amino acid sequence of SEQ ID NO: 194. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 195, or the amino acid sequence of SEQ ID NO: 195. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 196, or the amino acid sequence of SEQ ID NO: 196. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 197, or the amino acid sequence of SEQ ID NO: 197.In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 198, or the amino acid sequence of SEQ ID NO: 198. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 199, or the amino acid sequence of SEQ ID NO: 199. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 200, or the amino acid sequence of SEQ ID NO: 200. In some embodiments, the CAR of the present disclosure includes an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least ...

Claims

1. It is a chimeric antigen receptor (CAR), (a) an extracellular antigen-binding domain that specifically binds to the CD79b antigen, (b) Transmembrane domain and (c) Including an intracellular signaling domain, The extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and the light chain variable region includes an amino acid sequence that is at least 90% identical to SEQ ID NO:

32. A CAR wherein the heavy chain variable region includes a heavy chain complementarity determination region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 257, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 258, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 259, and the light chain variable region includes a light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 261, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

262.

2. The extracellular antigen-binding domain is The CAR according to claim 1, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

32.

3. The CAR according to claim 1 or 2, wherein the extracellular antigen-binding domain comprises a single-stranded variable fragment (scFv), and the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL).

4. The CAR according to claim 3, wherein the scFv includes a linker polypeptide between the heavy chain variable region (VH) and the light chain variable region (VL).

5. The CAR according to claim 4, wherein the linker polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

42.

6. The linker polypeptide comprises the amino acid sequence of SEQ ID NO: 42, as described in claim 4.

7. The CAR according to any one of claims 3 to 6, wherein the scFv includes an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 91 and 113.

8. The CAR according to any one of claims 3 to 7, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 91 and 113.

9. The CAR according to any one of claims 3 to 8, wherein the scFv comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

113.

10. The scFv comprises the amino acid sequence of SEQ ID NO: 113, as described in any one of claims 3 to 9.

11. The CAR according to any one of claims 1 to 10, wherein the extracellular antigen-binding domain comprises a signal polypeptide.

12. The CAR according to claim 11, wherein the signal polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

37.

13. The CAR according to claim 11, wherein the signal polypeptide comprises the amino acid sequence of SEQ ID NO:

37.

14. The CAR according to any one of claims 1 to 13, wherein the intracellular signal transduction domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3ζ chain (CD3z) component, a differentiation cluster (CD27) component, a differentiation cluster superfamily member component, and combinations thereof.

15. The CAR according to claim 14, wherein the CD137 component includes an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

40.

16. The CAR according to claim 14, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO:

40.

17. The CAR according to claim 14, wherein the CD3z component includes an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

41.

18. The CAR according to claim 14, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO:

41.

19. The CAR according to any one of claims 1 to 18, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

20. The CAR according to claim 19, wherein the CD8a-TM polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

39.

21. The CAR according to claim 19, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:

39.

22. The CAR according to any one of claims 1 to 21, further comprising a hinge region that links the transmembrane domain to the extracellular antigen-binding domain.

23. The CAR according to claim 22, wherein the hinge region is the CD8a hinge region.

24. The CAR according to claim 23, wherein the CD8a hinge region includes an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

38.

25. The CAR according to claim 23, wherein the CD8a hinge region includes the amino acid sequence of SEQ ID NO:

38.

26. The CAR is the CAR according to any one of claims 1 to 25, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 180 and 202.

27. The CAR according to any one of claims 1 to 26, wherein the CAR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

202.

28. The CAR is the CAR according to any one of claims 1 to 27, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

202.

29. Isolated lymphocytes expressing CAR according to any one of claims 1 to 28.

30. The isolated lymphocytes according to claim 29, wherein the lymphocytes are T lymphocytes.

31. The isolated lymphocytes according to claim 30, wherein the lymphocytes are natural killer (NK) cells.

32. An isolated nucleic acid molecule encoding a CAR according to any one of claims 1 to 28.

33. A vector comprising the nucleic acid molecule described in claim 32.

34. A cell expressing the nucleic acid molecule described in claim 32.

35. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of claims 29 to 31 and a pharmaceutically acceptable excipient.

36. A pharmaceutical composition according to claim 35, used for treating a subject having cancer.

37. The pharmaceutical composition according to claim 36, wherein the cancer is B-cell lymphoma.

38. The pharmaceutical composition according to claim 36, wherein the cancer is non-Hodgkin lymphoma.

39. The pharmaceutical composition according to claim 36, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasmacytoma.

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