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

Abstract

This invention provides a chimeric antigen receptor (CAR) that specifically targets the differentiated antigen group 79b protein (CD79b) for the treatment of cancer, and immune-responsive cells containing such CARs. [Solution] A chimeric antigen receptor (CAR) is provided, comprising (a) an extracellular domain that specifically binds to the CD79b antigen, (b) a transmembrane domain, and (c) an intracellular signaling domain that optionally includes at least one costimulatory domain.

Description

Technical Field

[0001] (Sequence Listing) This application has been electronically filed in ASCII format, and the entire sequence listing is incorporated herein by reference. The 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, which comprises a single-chain variable fragment targeting CD79b, and a genetically engineered immune cell expressing the CAR that targets CD79b. 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 the CAR that targets CD79b. Also provided are methods for producing the CAR and the genetically engineered immune cell, and methods for using the genetically engineered immune cell for treating a condition including cancer.

Background Art

[0003] In T cell therapy, genetically modified isolated T cells are utilized to enhance specificity for certain tumor-associated antigens. The genetic modification involves the expression of a chimeric antigen receptor (CAR) or an exogenous T cell receptor, which can provide a new antigen specificity on the T cell. T cells expressing a chimeric antigen receptor (CAR-T cells) can induce a tumor immune response. 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 treatments, relapsed / refractory (r / r) NHL remains uniform. Characterized by a poor prognosis. T14 is genetically modified to express chimeric antigen receptors (CARs). Cell-based adoptive immunotherapy has shown promising results in the treatment of CD19-positive B-cell malignancies. However, even if the initial overall response rate is around 60-80%, long-term complete remission may not be achieved. Only 40% of patients have this condition [1, 2]. Acute lymphoblastic leukemia emia (ALL) and diffuse large B-cell lymphoma Clinical studies report disease relapse due to CD19 antigen loss in both patients with DLBCL. New types of antigens are currently emerging that target novel surface antigens, addressing unresolved clinical needs. Its importance is attracting attention [2, 4].

[0005] B cells (B lymphocytes) are central components of adaptive immunity, producing antibodies and antigens. They perform the role of indicator cells, secrete cytokines, and develop into memory B cells after activation. B cells respond to several different pathogens.[5] B cells circulate in the blood and lymphatic system. In lymphoid organs, it encounters its own antigen, and additional signals are received from T helper cells. Both can differentiate into effector plasma cells. These cells circulate in the bloodstream. They secrete specific antibodies that target and eliminate antigens or pathogens.[6]

[0006] To detect antigens or pathogens, B cells have B cell receptors on their cell surface. It contains tor, BCR), which is a transmembrane immunoglobulin molecule (mIg), as well as CD79 A multicomponent consisting of a disulfide-bonded heterodimer of a(Igα) and CD79b(Igβ). It is a receptor. CD79b is highly expressed in a wide range of B-cell lymphomas. This has been shown to be important for cancer cell survival in most DLBCL tumor models. Therefore, the development of resistance to CD79b-targeted drugs due to antigen loss is unlikely. Therefore, it could be an attractive target for the development of novel immunotherapy approaches. In clinical practice, C Antibody-drug conjugates (ADCs) targeting D79b Polatuzumab (Polivy®), a derivative of this drug, has recently been used as a treatment for r / rDLBCL. It was approved for [7]. Polatuzumab treatment is an alternative to standard treatment (bendamustine and ritudium). Complete response (CR) and duration of response (dur) when combined with simab. The rate of response (DOR) increased, and CD79b was validated as a valuable clinical target. do [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] Chimeric antigen receptors (CARs), for example, the differentiated antigen group 79B protein (Cluster of Differentiated Antigens) CARs targeting retinosis 79B protein (CD79b), CAR-containing cells, CAR 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 Furthermore, methods for treating disorders, such as cancer, using the disclosed CAR are disclosed herein. It will be shown.

[0009] In one embodiment, a chimeric antigen receptor (CAR) is provided, comprising the following: (a) scFv, which specifically binds to the differentiated antigen group 79B protein (CD79b) antigen. An extracellular domain including, (b) Transmembrane domain and (c) Intracellular signaling domains 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 in question is a TNF receptor superfamily member 9(C) The costimulatory domain containing component D137 and the T cell surface glycoprotein CD3ζ chain (CD3z It contains a primary signaling domain that includes the component.

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

[0012] In another embodiment, the extracellular antigen-binding domain, the transmembrane domain, and the intracellular signaling domain A chimeric antigen receptor (CAR) including the main, wherein the extracellular antigen-binding domain is Heavy chain complementarity determination region 1 having the amino acid sequence of SEQ ID NO: 208 The mining region (CDR1), the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 209, and 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, amino acid sequence of SEQ ID NO: 217 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 218, and a 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, amino acid sequence of SEQ ID NO: 223 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 224, and a 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, amino acid sequence of SEQ ID NO: 217 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 229, and a 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, amino acid sequence of SEQ ID NO: 233 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 234, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 238, amino acid sequence of SEQ ID NO: 239 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, and a 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, amino acid sequence of SEQ ID NO: 243 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 244, and a 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, amino acid sequence of SEQ ID NO: 249 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 250, and a 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, amino acid sequence of SEQ ID NO: 254 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 255, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 257, amino acid sequence of SEQ ID NO: 258 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 259, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 263, amino acid sequence of SEQ ID NO: 243 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 264, and a 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, amino acid sequence of SEQ ID NO: 269 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 270, or Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 274, amino acid sequence of SEQ ID NO: 275 It includes a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 276 and a 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, the extracellular antigen-binding domain, the transmembrane domain, and the intracellular signaling domain A chimeric antigen receptor (CAR) including the main, wherein the extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, amino acid sequence of SEQ ID NO: 220 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 231 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 246 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 252, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 256, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 262, and a 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, amino acid sequence of SEQ ID NO: 266 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, amino acid sequence of SEQ ID NO: 272 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 273, or Light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, amino acid sequence of SEQ ID NO: 266 It includes a light chain CDR2 having the amino acid sequence of SEQ ID NO: 278 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, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, amino acid sequence of SEQ ID NO: 220 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 231 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 246 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 252, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 256, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 262, and a 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, amino acid sequence of SEQ ID NO: 266 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, amino acid sequence of SEQ ID NO: 272 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 273, or Light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, amino acid sequence of SEQ ID NO: 266 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 278, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 278, further Includes. In another embodiment, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor (CAR) is provided, including the extracellular antigen-binding domain. Sequence numbers 208, 216, 222, 228, 232, 238, 242, 248, 253 Heavy chain CDR1 having amino acid sequences of 257, 263, 268, or 274, SEQ ID NO: 209, 217, 223, 233, 239, 243, 249, 254, 258, 269, Alternatively, heavy chain CDR2 having a 275-amino acid sequence, SEQ ID NOs: 210, 218, 224, 2 29, 234, 240, 244, 250, 255, 259, 264, 270, or 276 Heavy chain CDR3 having the amino acid sequence, SEQ ID NOs: 211, 214, 215, 219, 22 5, 230, 235, 241, 245, 251, 260, 265, 271, or 277 Light chain CDR1 with amino acid sequence, SEQ ID NOs: 212, 220, 226, 231, 236 , light chain CDR2 having an amino acid sequence of 246, 261, 266, or 272, and sequence Numbers 213, 221, 227, 237, 247, 252, 256, 262, 267, 27 It contains a light chain CDR3 having an amino acid sequence of 3 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) Amino acids of sequence numbers 274, 275, 276, 277, 266, and 278, respectively. Acid sequence, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CD Includes R2 and light chain CDR3.

[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 sequence number 1, and sequence Light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 1, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 2, and sequence Light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 2, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 4, and sequence Light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 5, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 22, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 5, and sequence Light chain variable region containing an amino acid sequence that is at least 90% identical to number 23, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 6, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 24, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 7, and sequence Light chain variable region containing an amino acid sequence that is at least 90% identical to number 26, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 8, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 25, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 9, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 27, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 28, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 29, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 30, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 31, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 32, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 33, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 34, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 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 sequence number 33, or A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, and It contains a light chain variable region that includes an amino acid sequence that is at least 90% identical to that of column number 36.

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

[0018] 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: 14, and It contains a light chain variable region that includes an amino acid sequence that is at least 90% identical to that of column number 32.

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

[0020] In some embodiments, the extracellular antigen-binding domain is a single-chain variable fragment. Includes a variable fragment (scFv). In some embodiments, scFv is a light chain variable. It contains a linker polypeptide between the variable region (VL) and the heavy chain variable region (VH). In this embodiment, the linker polypeptide is at least 90% identical to SEQ ID NO: 42. It contains a mino acid sequence. In some embodiments, the linker polypeptide is the same as in SEQ ID NO: 42 It contains an amino acid sequence. In some embodiments, scFv is derived from SEQ ID NOs. 75-118. It contains amino acid sequences that are at least 90% identical to sequences selected from the group. In this embodiment, scFv is an amino acid selected from the group consisting of SEQ ID NOs. 75 to 118. It contains an acid sequence. In some embodiments, the extracellular antigen-binding domain is a signaling polypeptide. Contains cydo. In some embodiments, the signal polypeptide is at least SEQ ID NO: 37 It also contains amino acid sequences that are 90% identical. In some embodiments, signal peptides D contains the amino acid sequence of SEQ ID NO: 37.

[0021] In some embodiments, the intracellular signaling domain is a TNF receptor superfa Millie member 9 (CD137) component, T cell surface glycoprotein CD3ζ chain (CD3z) Components, differentiation cluster (CD27) components, differentiation cluster superfamily member components, It includes polypeptide components selected from the group consisting of combinations thereof. In this embodiment, component CD137 is an amino acid compound that is at least 90% identical to that of SEQ ID NO: 40. Includes a column. In some embodiments, component CD137 has the amino acid sequence of SEQ ID NO: 40 Includes. In some embodiments, the CD3z component is at least 90% identical to SEQ ID NO: 41. It contains a certain amino acid sequence. In some embodiments, the CD3z component is the A of SEQ ID NO: 41 It contains a mino acid sequence. In some embodiments, the intracellular signaling domain is SEQ ID NO: 1 It contains an amino acid sequence that is at least 90% identical to 63. In some embodiments, the cell The internal signaling domain contains the amino acid sequence of SEQ ID NO: 163. Several embodiments Therefore, the transmembrane domain contains the CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide is at least 90 of SEQ ID NO: 39. Contains % identical amino acid sequences. In some embodiments, CD8a-TM polypeptide D contains the amino acid sequence of SEQ ID NO: 39.

[0022] In various embodiments, the CAR ligates its transmembrane domain to an extracellular antigen-binding domain. Further includes a hinge region. In some embodiments, the hinge region is the CD8a hinge region. Yes. In some embodiments, the CD8a hinge region is at least 90% of Sequence ID No. 38. It contains the same amino acid sequence. In some embodiments, the CD8a hinge region is sequence number Contains amino acid sequence 38.

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

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

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

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

[0027] In some embodiments, the extracellular antigen-binding domains are sequence numbers 257 and 25, respectively. Heavy chain CDR1 and heavy chain CD have amino acid sequences of 8, 259, 260, 261, and 262. It may include R2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.

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

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

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

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

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

[0033] In some embodiments, the intracellular signaling domain is a TNF receptor superfa Millie member 9 (CD137) component, T cell surface glycoprotein CD3ζ chain (CD3z) Components, differentiation cluster (CD27) components, differentiation cluster superfamily member components, It may also include polypeptide components selected from the group consisting of combinations thereof. As a typical example, component CD137 is an amino acid that is at least 90% identical to that of SEQ ID NO: 40. It may contain sequences. The CD3z component is an amino acid that is at least 90% identical to that of SEQ ID NO: 41. The sequence may contain an intracellular signaling domain that is at least 90% identical to that of Sequence ID No. 163. It may contain the amino acid sequence. As another non-limiting example, component CD137 is sequence number It may contain 40 amino acid sequences. The CD3z component may contain the amino acid sequence of SEQ ID NO: 41. The intracellular signaling domain may contain the amino acid sequence of SEQ ID NO: 163.

[0034] In certain embodiments, the intracellular signaling domain is sequence number 163 and at least 90 It may contain amino acid sequences that are % identical. In certain embodiments, intracellular signaling domain The molecule may contain the amino acid sequence of sequence number 163.

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

[0036] In some embodiments, the CARs disclosed herein have a transmembrane domain that acts as an extracellular antigen It may further include hinge regions that connect to the binding domain. In certain embodiments, the hinge regions are , which may be the CD8a hinge region. In some embodiments, the CD8a hinge region is an array It may include an amino acid sequence that is at least 90% identical to number 38. Such embodiments Therefore, the CD8a hinge region may contain the amino acid sequence of SEQ ID NO: 38.

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

[0038] In some embodiments, the CAR disclosed herein is sequence number 202 and at least 9 It may contain amino acid sequences that are 0% identical. In some embodiments, as disclosed herein The CAR may contain the amino acid sequence of SEQ ID NO: 202. In another embodiment, the above CAR Isolated lymphocytes expressing either are provided. In some embodiments, lymphocytes These are T lymphocytes. In some embodiments, lymphocytes are natural killer (NK) cells. ) These are cells.

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

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

[0041] In another embodiment, a method is provided for treating a subject having cancer. The method is therapeutically effective Administer any of the above-mentioned lymphocytes or the above-mentioned pharmaceutical composition in the amount required to a subject in need. This includes the process by which lymphocytes induce the killing of cancer cells in the target. In some embodiments, the cancer is B-cell lymphoma. This is a non-Hodgkin lymphoma. In some embodiments, the cancer is diffuse large cell type B DLBCL (Deutzia Cellular Lymphoma), Mantle Cell Lymphoma (MCL) follicular lymphoma (FL), marginal zone lymphoma phoma, MZ), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (chronic lymphocytic leukemia) CLL (Chronic leukemia), multiple myeloma (MM), mucosal-associated lymphoma Tissue lymphoma (mucosa-associated lymphoid tissue, MALT), Hodgkin lymphoma, It is Burkitt lymphoma, hairy cell leukemia, or plasmacytoma.

[0042] In another embodiment, a method for targeted death of cancer cells is provided, wherein the method involves killing cancer cells as described above. This involves contacting one of the lymphocytes, thereby causing the lymphocytes to kill the cancer cells. Induce. In some embodiments, cancer cells are malignant B cells. In this embodiment, cancer cells are non-Hodgkin lymphoma cells. In some embodiments, cancer The cells are used in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MC). L), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia (ALL) ), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), mucosa-associated lymphoid tissue (MA) LT) Lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasma cell leukemia These are cells from a cytoma.

[0043] In another embodiment, a method for detecting the presence of cancer in a subject, (a) By bringing a cell sample obtained from the subject into contact with any of the above CARs, C To form an AR-cell complex, (b) Detecting the complex (where the detection of the complex indicates the presence of cancer in the subject) A method is provided that includes ( ). [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. It will probably happen.

[0045] This patent application document includes at least one color drawing. A copy of this patent application having (possible) may be submitted by the Japan Patent Office upon request and payment of the necessary fees. It will be provided. [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 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 relates to a chimeric antigen receptor that targets the differentiated antigen group 79B protein (CD79b). (CAR), cells containing such CARs, and using the CARs described herein, To provide a method for treating (for example, B-cell lymphoma).

[0048] The CAR of the present invention has antigen specificity for CD79b. And the phrase "induces an antigen-specific response" means, when used herein, that the CAR is an antigen. It can specifically bind to and immunologically recognize the CD79b antigen of CAR. This means that binding to the antigen triggers an immune response. Antigen specificity and recognition of target cells Methods for testing CAR's ability are known in the art.

[0049] This disclosure also relates to relevant nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies, or The antigen-binding portion and pharmaceutical compositions relating to the CAR of the present invention are also provided.

[0050] Some aspects of the present invention are described below with reference to examples provided for illustrative purposes only. To provide a complete understanding of the present invention, numerous specific details, relationships, and methods are described. Please understand that this is the case. However, those skilled in the art will know that the present invention is not a concrete detail of the present invention. It must be possible to carry out the procedure without one or more of the following, or by other methods, protocols, or reagents. It will be readily apparent that this can be done in cell lines and animals. Because the actions may occur in a different order and / or in parallel with other actions or events, The explanation is not limited by the order of the actions or events exemplified. Furthermore, the method according to the present invention Not all of the exemplified actions, steps, or events are necessarily required for implementation. i. Many of the techniques and procedures described or referenced herein are by those skilled in the art. It is well understood and commonly used with conventional methods.

[0051] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein , or technical terms, have meanings that are ordinarily understood by those skilled in the art to whom this invention relates. Intention. In some cases, terms that have a commonly understood meaning are used for clarity. and / or as defined herein for immediate reference, including such definitions herein. To do so will always reveal a substantial difference from what is generally understood in the relevant technical field. It should not be interpreted in any way. Terms such as those defined in commonly used dictionaries, As having meanings consistent with those meanings in the context of related technologies, and / or this It is further understood that it needs to be interpreted as being defined otherwise in the specification. It will probably happen.

[0052] definition When a list is presented, unless otherwise specified, each individual element of that list and its list It should be understood that all combinations of strikes are distinct embodiments. For example, "A, The list of embodiments presented as "B" or "C" is, It is interpreted as including "A or B", "A or C", "B or C", or "A, B, or C". It should be done.

[0053] The terms used herein are for the sole purpose of describing specific embodiments. This is not intended to be an exhaustive limitation. When used herein, it is clearly indicated otherwise depending on the context. Unless otherwise specified, the articles "a," "an," and "the" are understood to refer to multiple things. I want to be treated that way.

[0054] Transition phrases: "comprising," "consisting essential" The terms "ly of" and "consisting" are generally accepted in patent terminology. It is intended to imply the meaning of "to be equipped / include (compri "Sing" is synonymous with "include," "contain," or "characterize," and is comprehensive or non-comprehensive. This is restrictive and does not exclude other elements or methods not listed. (ii) "consisting of" is specified in the claims. (iii) "essentially consisting of" This refers to the specified materials or processes, as well as the "basic and novel features" of the claimed invention. The scope of the patent claims is limited to those that do not substantially affect (multiple) the patent. Embodiments described in relation to "comprising" (or equivalent) are also implementations. As a matter of state, I propose to present independently the descriptions of "consisting of" and "essentially becoming from". To provide.

[0055] The terms "approximately" or "about" include the fact that the value falls within a statistically meaningful range. Such a range is within one decimal place of a given value or range, preferably within 50%, more preferably within 2. It may be within 0%, more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the words "approximately" or "about" is a specific system under study. It depends on the element and can be easily understood by those skilled in the art.

[0056] "Activation" or "stimulation" means that cells (e.g., T cells and NK cells) are activated as a result. To express sexualization markers, produce cytokines, proliferate, and / or target cells This means inducing a change in the biological state of the cell, which can lead to cytotoxicity. These changes are all This can be caused by the primary stimulus signal. The co-stimulatory signal increases the magnitude of the primary signal. This broadens the range of cells and suppresses cell death after initial stimulation, resulting in a more durable activated state. This leads to a state and, consequently, higher cytotoxicity. "Co-stimulatory signaling" is TCR / CD3 In combination with primary signaling such as eruption, T cell and / or NK cell proliferation And / or upregulation or downregulation of key molecules It refers to the signal that is triggered.

[0057] "Bispecificity" refers to the ability to identify two different antigens or two different epitopes within the same antigen. This refers to molecules that specifically bind (e.g., antibodies). Bispecific molecules bind to other related antigens. For example, humans or monkeys, for example, crab-eating macaques (Macaca cynomolgus) (for example, crab The same antigen (homolo) is derived from other species such as izal (cynomolgus, cyno) or chimpanzees. It may have cross-reactivity with (g), or it may be shared between two or more different antigens. It can bind to epitopes.

[0058] The terms "chimeric antigen receptor" or "CAR" are used herein in reference to all naturally occurring substances. A combination not found together in a single protein, extracellular target binding domain It is defined as a cell surface receptor containing a transmembrane domain and an intracellular signaling domain. This is understood to mean that, in particular, the extracellular domain and intracellular signaling domain are naturally The present invention includes receptors that are not found together in a single receptor protein. Chimeric antigen receptors are mainly found in lymphocytes such as T cells and natural killer (NK) cells. It is intended for use.

[0059] The term "coding" refers to a specific sequence of nucleotides (e.g., rR A biological organism having either a specific sequence of amino acids (NA, tRNA, and mRNA) or amino acids. It serves as a template for the synthesis of other polymers and macromolecules in scientific processes. The specificity of nucleotides in polynucleotides such as genes, cDNA, or mRNA. This refers to the inherent characteristics of a specific arrangement, as well as the biological characteristics arising therefrom. Therefore, cells or In other biological systems, the transcription and translation of mRNA corresponding to genes produce proteins. In this case, the gene, cDNA, or RNA codes for a protein. A coding strand whose columns are identical to the mRNA sequence, and a transcription template for a gene or cDNA. Any non-coding strand used in this way is a protein or other part of the gene or cDNA. It can be mentioned as encoding a product.

[0060] Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" are mutually degenerate. All nucleotide sequences that are of the same type, and all nucleotide sequences that encode the same amino acid sequence. To include. The phrase "nucleotide sequence encoding a protein or RNA" also includes several. In that case, the nucleotide sequence encoding the protein may contain introns. It can encompass introns to a certain extent.

[0061] The term "expression vector" refers to a vector that can manipulate the nucleotide sequence to be expressed. This refers to a vector containing recombinant polynucleotides that include a linked expression regulatory sequence. The gene contains sufficient cis-acting elements for expression, and other elements for expression are present in the host cell. It can be supplied by or in an in vitro expression system. The expression vector is recombinant Cosmids and plasmids incorporating polynucleotides (for example, naked or liposomes) those contained), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) include all vectors known in the art.

[0062] "Heterologous" refers to two or more polynucleotides or two or more polypeptides that are not found in nature in the same relationship to each other.

[0063] "Heterologous polynucleotide" refers to a polynucleotide that does not occur naturally and encodes two or more neoantigens described herein.

[0064] "Heterologous polypeptide" refers to a polypeptide that does not occur naturally and contains two or more neoantigen polypeptides described herein.

[0065] "Host cell" refers to any cell that contains heterologous nucleic acid. Exemplary heterologous nucleic acids are vectors (e.g., expression vectors).

[0066] The terms "T cell" and "T lymphocyte" are interchangeable and used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL, CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL, CD8+ T cells), tumor infiltrating lymphocyte cytotoxic T cells (TIL, CD8+ T cells), CD4+CD8+ T cells, or any T cells. ​​​​​Other subsets may also be used. Other examples of T cells suitable for use in a particular embodiment Symbolic populations include naive T cells and memory T cells. Also, "NKT cells" This also includes cells that express semi-invariant αβ T cell receptors, as well as NK1.1, etc. This refers to a special group of T cells that also express various molecular markers typically associated with NK cells. KT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, and CD8 Examples include + and CD8 cells. The TCR of NKT cells is the MHC I-like molecule CD Id It is unique in that it recognizes glycolipid antigens presented by NKT cells. It has the ability to produce cytokines that promote either immune tolerance or harmful effects. It may have any of the following effects. It also includes "gamma delta T cells (γδ T cells)." Gamma delta T cells are a small subset of T cells that have different TCRs on their surface. This refers to a special group of individuals, where TCR is represented as two glycoprotein chains, α and β-TCR chains. Unlike most T cells, which are composed of the gamma and delta chains, the TCR in γδ T cells is different. It is composed of the following. γδT cells can play a role in immune surveillance and immunomodulation. It is an important source of IL-17 and induces an active CD8+ cytotoxic T cell response. It was found that this suppresses abnormal or excessive immune responses and plays a role in immune tolerance. This also includes "regulatory T cells" or "Treg," which refer to T cells that perform a specific function. Treg cells are a classic example. In terms of type, it is a transcription factor Foxp3-positive CD4+ T cell, and also an IL-10-producing CD4+ T cell. This can also include Foxp3-negative regulatory T cells, which are +T cells.

[0067] The terms "natural killer cell" and "NK cell" are interchangeable and used herein. Used synonymously. In this specification, NK cells refer to CD16+CD56+ and NK refers to differentiated lymphocytes that have a CD57+TCR- phenotype. Activation of the detoxifying enzyme binds to cells that cannot express "self" MHC / HLA antigens, killing them. The ability to injure, kill tumor cells or other disease cells that express ligands for NK cell activation receptors. The ability to release protein molecules called cytokines that stimulate or inhibit the immune response. It is characterized by the ability to do so.

[0068] As used herein, the term “antigen” means an antigen to which a T cell receptor can bind. Ingredients (for example, proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleic acids, etc.) It refers to a molecule (part of or a combination thereof). Antigens can also trigger an immune response. Examples of immune responses include, but are not limited to, antibody production or specific immunological conditions. This may involve activation of pitent cells, or both. Those skilled in the art will know that the antigen never becomes a "gene". Therefore, you will understand that it does not need to be coded. Antigens are produced by synthesis. It may also be derived from a biological sample, or it may be a macromolecule other than a polypeptide. It is readily apparent that this is acceptable. Such biological samples include tissue samples and tumor samples. Cerebral samples, cells, or other biological components, organisms, protein / antigen subunits, killing or Alternatively, fluids containing inactivated whole cells or lysates can be cited, but these Not limited to this.

[0069] An "antigen-binding fragment" or "antigen-binding domain" is a part of a protein that binds to an antigen. Refers to. The antigen-binding domain may be a synthetic polypeptide, an enzymatically available polypeptide, or a genetically engineered polypeptide, including a part of an immunoglobulin that binds to an antigen, such as VH, VL, VH and VL, Fab, Fab’, F(ab’)2, F d and Fv fragments, a domain antibody (d Ab) consisting of one VH domain or one VL domain, a shark variable IgNAR domain, a camelized VH domain, a VHH domain, an FR3 -CDR3-FR4 moiety, etc., a minimum recognition unit consisting of amino acid residues that mimic the CDR of an antibody, such as HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR 2, and / or LCDR3, an alternative scaffold that binds to an antigen, and a multispecific protein containing an antigen-binding fragment. Antigen-binding fragments (such as VH and VL) are linked to each other via a synthetic linker so that when the VH and VL domains are expressed by separate single chains, the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain , for example, a single-chain Fv (scFv) or a diabody, and various types of single-chain antibody designs can be formed. Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds that can be monospecific or multispecific for genetic engineering of bispecific and multi specific proteins. The term "antibody" refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chi meric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, dis ulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-i mmunoglobulins, etc.

[0070] The term "antibody" includes 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-immunoglobulins, etc. ulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-i Diotype (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), This refers to either the antibody or any of the epitope-binding fragments mentioned above. The terms "antibody" and "antibody" are also used. Covalent dies, such as those described in U.S. Patent Application Publication No. 2007 / 0004909. Abody, and those disclosed in U.S. Patent Application Publication No. 2009 / 0060910 Which Ig-DARTS are you referring to? Antibodies useful as TCR-binding molecules include immunoglobulins. It contains immunologically active fragments of immunoglobulin molecules and immunoglobulin molecules, i.e., antigen-binding sites. Examples of such molecules include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG) 3. IgG4, IgM1, IgM2, IgA1, and IgA2), or subclasses That's fine.

[0071] Human antibodies are optimized to produce the minimum possible immune response when administered to human subjects. This refers to antibodies that have been modified. The variable region of human antibodies is derived from the human immunoglobulin sequence. If the antibody includes a constant region or a part of a constant region, the constant region is also human immunoglobulin Derived from the row. Human antibodies are derived from human germline immunoglobulins or rearranged in the variable region of human antibodies. When obtained from a system using the immunoglobulin gene, the sequence is of human origin. This includes a heavy chain variable region and a light chain variable region. Such exemplary systems are used to phages. A sprayed human immunoglobulin gene library and human immunoglobulin gene loci. This includes transgenic non-human animals possessing "human antibodies," such as mice or rats. " is typically a system used to obtain human antibodies and human immunoglobulin gene loci. The difference lies in the intentional introduction of somatic mutations into the framework or CDR. By means of, or both of these, the amino acid content when compared to immunoglobulins expressed in humans It contains differences. Typically, "human antibodies" are human germline immunoglobulins or reorganized immunoglobulins. For the amino acid sequence encoded by the immunoglobulin gene, The sequence is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, and 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% They are 98% or 99% identical. In some cases, "human antibodies" are, for example, Knappi k et al., (2000) J Mol Biol 296:57-86 Consensus framework sequences obtained from human framework sequence analysis, or for example Shi et al., (2010) J Mol Biol 397:385-96 and The human immune phages presented in International Publication No. 2009 / 085462 It may contain synthetic HCDR3 incorporated into a globulin gene library. Antibodies in which one of the CDRs originates from a non-human species are not included in the definition of "human antibodies."

[0072] "Humanized antibodies" are those in which at least one CDR is derived from a non-human species and at least one F The framework refers to antibodies derived from human immunoglobulin sequences. Humanized antibodies are frameworks. Because the framework can include substitutions, the framework expresses human immunoglobulins. It may not be an exact copy of the germline gene sequence of phosphorus or human immunoglobulin. .

[0073] "Specifically bind," "specifically bind," "specifically bound," or "to bind" "Specific binding" 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 has an equilibrium dissociation constant (K d) of about 1× 10 , , , D , , ,

[0074] 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 ) to bind to the antigen or an epitope within the antigen, and typically, K D is at least 100-fold less than K D for binding to non-specific antigens (e.g., BSA, casein). In the context of the CD79b antigen described herein, "specific binding" D 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. The term "host cell" means any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector - (e.g., an expression vector). For example, a host cell can be any cell of biological origin that is selected, modified, transformed, grown, used, or manipulated in any way for the production of a substance by the cell, e.g., for the expression in the cell of a gene, DNA or RNA sequence, protein, or enzyme. A suitable host can be determined. For example, a host cell can be selected based on the vector

[0074] backbone and the desired result. By way of example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate some types of vectors. DH - ​​​​​Bacterial cells such as 5α, JM109, and KCB, but not limited to these, SURE (Registered Trademark) Competent cells, as well as SOLOPACK Gold cells, are vector-compounded. It can be used as a host cell for production and / or expression. Furthermore, Escherichia coli LE39 Bacterial cells such as those listed in 2 can be used as host cells for phage viruses. Eukaryotic cells that can be used as cells include yeast (e.g., YPH499, YPH500 and YPH499). This includes, but is not limited to, PH501), insects, and mammals. Vector replication Examples of mammalian eukaryotic host cells for and / or expression include HeLa, NIH3T3, J Examples include urkat, 293, COS, CHO, Saos, and PC12, but these Not limited to this.

[0075] The host cells of this disclosure contain a DNA or RNA sequence encoding a CAR. Examples include T cells and natural killer cells that express CAR on their cell surface. (Host cell) It increases T cell activity, natural killer cell activity, cancer treatment, and treatment of autoimmune diseases. It can be used to strengthen.

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

[0077] The term "differentiation" refers to reducing the ability or proliferation of a cell, or limiting the cell's developmental capabilities. This refers to a method of transitioning to a predetermined state.

[0078] The terms "express" and "expression" refer to the generation of information about a gene or DNA sequence. To enable or generate, for example, the transcription and translation of a corresponding gene or DNA sequence. This means generating proteins by activating cellular functions involved in D NA sequences are expressed within or by cells to form "expression products" such as proteins. The expression product itself, for example, the resulting protein, is said to have been "expressed" by the cell. It is also possible to characterize the expression product as intracellular, extracellular, or transmembrane. ru.

[0079] The term "transfection" refers to the use of recombinant DNA technology to "external" (i.e., This refers to introducing exogenous or extracellular nucleic acids into cells. The term refers to a host cell expressing an introduced gene or sequence to produce a desired substance, usually introduced. "Foreign" genes or sequences are used to produce proteins or enzymes encoded by the genes or sequences. The introduction of (exogenous or extracellular) genes, DNA, or RNA sequences into host cells. This means that the introduced gene or sequence is also a "cloned" or "foreign" gene. They may also be called sub-sequences or sequences, and are starters, terminaters, promoters, signals, secretions, or sub-sequences. Poly, which encodes chimeric antigen receptors, such as other sequences used by the genetic mechanism of cells. It may contain a regulatory or control sequence that is manipulably ligated to a nucleotide. The sequence may include non-functional sequences that do not have known functions. The introduced DNA or R The host cells that receive and express NA are "genetically engineered". The extracted DNA or RNA may originate from any source, including cells of the same genus or species as the host cell, or from a different source. It may originate from a genus or species.

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

[0081] The term "regulatory element" refers to any systolic function that controls several aspects of nucleic acid sequence expression. It refers to a functional genetic element. In some embodiments, the term "promoter" essentially means a transforming genetic element. Includes the minimum sequence required to start the copy. In some embodiments, the term "promoter" is used. The "-" contains a sequence to initiate transcription, and in addition, each is generally referred to as an "enhancer". Upregulate or This also includes sequences that can be downregulated.

[0082] As used herein, “operable connection” or “operable connection” The terms "did" and similar phrases, when used in relation to nucleic acids or amino acids, each This refers to a manipulable linkage of nucleic acid sequences or amino acid sequences that are in a functional relationship with one another. For example, a promoter, enhancer element, or an operable, linked together The pun-reading frame, 5' and 3' UTR, and terminator sequence are nucleic acid components. This results in the precise production of offspring (e.g., RNA). In some embodiments, it is operable. The nucleic acid elements linked in this manner are used for the transcription and final processing of the open reading frame. This leads to polypeptide generation (i.e., expression of the open reading frame). As another example, peptides linked in a manipulable manner have functional domains that are compatible with each other. This refers to elements that are positioned at a close distance to each other, providing them with the intended functions of their respective domains.

[0083] The terms "to treat" or "treatment" do not mean delaying an unwanted physiological change or disease. To reduce, or to achieve beneficial or desired clinical outcomes during treatment. This refers to a therapeutic procedure whose purpose is to achieve a beneficial or desired clinical outcome, such as a detectable or Whether undetectable or not, it can alleviate symptoms, reduce the severity of the disease, and stabilize ( In other words, a disease state that does not worsen, a delay or slowing of disease progression, or improvement or alleviation of the disease state. , and remission (whether partial or complete) are also included. "Treatment" is Furthermore, the survival time was extended compared to the expected survival time if the subjects had not received treatment. This could mean causing unwanted physiological changes or Subjects who already have a disease, as well as subjects who have physiological changes or a tendency to develop a disease It is included.

[0084] As used herein, the term "subject" refers to an animal. The term "subject" may be used interchangeably herein with respect to the subject. "Target" includes people who are treated as patients for a disease or for the prevention of a disease. The methods described in the details may be used to treat animal subjects belonging to any classification. Examples of animals that fit this description include mammals. Examples of mammals include mice and hamsters. Examples include rodents such as [name of rodent] and lagomorphs such as rabbits, but this list is not limited to these. No. Mammals are descended from the order Carnivora, which includes felines (cats) and canines (dogs). It could be. Mammals include Bovidae (cattle) and Suidae (pigs). They may be from the order Artiodactyla, or the order Perissodactyla, which includes the family Equidae (horses). Mammals include monkeys. Order Ceboid, or Simoid (monkeys), or suborder Haplorhini (H) It could be a toe or an ape. In one embodiment, the mammal is a human.

[0085] "Enhancement," "promotion," "increase," "expansion," or "improvement" generally refer to a vehicle. Or a greater response compared to the response caused by either of the control molecules / compositions. To produce, induce, or cause a physical response (i.e., a downstream effect), This refers to the capabilities of the composition intended in the details. Measurable physiological responses are particularly relevant to the art and As is clear from the understanding of the description herein, T cell expansion, activation, effector function, This may include increased persistence and / or increased cancer cell killing ability. In certain embodiments, The amount that is "increased" or "enhanced" may be a "statistically significant" amount, as described in 1.1, 1.2. 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more. An increase of (for example, 500, 1000 times) (intermediate and greater than 1, for example, 1.5, 1. It may include all integers and decimals such as 6, 1.7, 1.8, etc. The response is vehicle Alternatively, it is produced by a control composition.

[0086] "Decrease" or "Lower" or "Make less" or "Reduce" or "Weaken" "Responding" generally refers to a response caused by either the vehicle or the control molecule / composition. Compared to that, it produces or induces fewer physiological responses (i.e., downstream effects), This refers to the ability of the composition intended herein to cause or result in. In certain embodiments, "reduce" The amount that is "reduced" or "decreased" may be a "statistically significant" amount, 1.1, 1.2, 1 0.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (example) For example, a decrease of 500, 1000 times (intermediate and greater than 1, e.g., 1.5, 1.6, Vehicles (1.7.1.8, etc.), response (reference response) produced by a control composition, or special This may include responses in specific cell lineages.

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

[0088] "Recurrent" means that the disease or signs and symptoms of the disease recur after a period of improvement following prior treatment with medication. It refers to resuming something.

[0089] "Intractable" refers to a disease that does not respond to treatment. Intractable diseases are diagnosed before treatment or at the start of treatment. Diseases that may be resistant to treatment or are intractable may become resistant to treatment during treatment.

[0090] "Tumor cells" or "cancer cells" refer to cells in vivo, ex vivo, or tissue culture. In any of the following cases, cancer or precancerous conditions exhibiting spontaneously occurring or induced phenotypic changes. This refers to sex-changed or transformed cells. These changes do not necessarily involve the uptake of new genetic material. Transformation is not the result of infection with a transformed virus and the incorporation of a new genomic nucleic acid. It can also be generated by the incorporation of exogenous nucleic acids, spontaneously or through exposure to carcinogens. It can develop after exposure, and in some cases, this can lead to mutations in endogenous genes. Transformation / cancer This involves morphological changes in vitro, in vivo, and ex vivo. Cell immortalization, abnormal growth control, formation of lesions, growth, malignant transformation, regulation of tumor-specific marker levels are exemplified by, for example, tumor growth in suitable animal hosts such as nude mice, through cell immortalization, abnormal growth control, formation of lesions, growth, malignant transformation, regulation of tumor-specific marker levels and the like.

[0091] The term "effective" as applied to a dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to bring about a desired activity when administered to a subject that requires it<00S1196>at the time of administration. When administering a combination of active ingredients, it should be noted that the effective amount of the combination may or may not include the amounts of each component that would be effective if administered individually. The exact necessary amount varies depending on the subject, depending on factors such as the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug used, the mode of administration, and the like.

[0092] The terms "prevent", "preventing", "prevention", or [ "prophylaxis" when used interchangeably in this specification are intended to mean preventing the disorder from occurring in the subject

[0093] The terms "therapeutically effective amount" or "effective amount" used interchangeably herein refer to an amount effective to obtain the desired therapeutic result at the required dose and duration. A therapeutically effective amount 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 status, reduction in tumor mass

[0094]

[0095] <00S1227>

[0096]

[0097] <00S1241>

[0098]

[0099] The phrase "pharmaceutically acceptable" as used in relation to the compositions described herein means Physiologically tolerable and usually harmful when administered to mammals (e.g., humans). This refers to molecular entities that do not undergo a reaction and other components of such compositions. Preferably, the term "medical "Pharmacologically acceptable" means that for use in mammals, more specifically in humans, it has been approved by the federal government. Alternatively, it may be authorized by a state government regulatory agency, or it may be regulated by the United States Pharmacopeia or other general regulations. It means that it is listed in the officially recognized pharmacopoeia.

[0095] The term "protein," as used herein, includes protein fragments of all lengths. In addition to natural and synthetic proteins, fusion proteins, and glycoproteins, all other Types of modified proteins (e.g., phosphorylation, acetylation, myristoylation, palmitoylation) Formulation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosyl Modifications (including, but not limited to, proteins obtained from pegylation, biotinylation, etc.) It encompasses all types of proteins.

[0096] Unless otherwise specified, the terms "nucleic acid," "nucleotide," and "polynucleotide" are used. It includes both DNA and RNA. "Nucleic acid sequence" or "nucleotide sequence" refers to a It refers to the nucleic acid sequence that codes for amino acids, and these terms are also coded by linkers. A cloning artifact that includes any amino acid being cloned. It can also refer to a nucleic acid sequence that contains the portion coding for a specific amino acid.

[0097] "Isolated" means that the molecule is not a component of the system in which it is produced, such as in recombinant cells. A homogeneous collection of molecules that have been substantially separated and / or purified (e.g., synthetic polynucleotides) In addition to oxides or polypeptides, tans subjected to at least one purification or isolation step This refers to a protein. "Isolated" means that it is substantially free from other cellular material and / or chemical substances. This refers to molecules that are not included, and indicates a higher purity, for example, 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Molecules isolated to a purity of 95%, 96%, 97%, 98%, 99%, or 100% are enclosed. Includes.

[0098] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle to which a compound is administered together. This refers to petroleum, animal, and plant-based medicinal carriers such as peanut oil, soybean oil, mineral oil, and sesame oil. Sterilizing liquids such as water and oil, including substances or synthetic materials. Water or aqueous solution, Physiological saline, as well as aqueous solutions of dextrose and glycerin, are used as carriers, especially for injection solutions. It is preferably used as a carrier, binder (for compressed pills), flow promoter, sealant. A solid dosage form carrier containing, but not limited to, one or more of the following: fillers, flavoring agents, and coloring agents. This may also be the case. An example of a suitable pharmaceutical carrier is "Reming" by EW Martin. It is listed in "ton's Pharmaceutical Sciences".

[0099] "Differentiation antigen group 79B protein" or "CD79b" refers to a B cell-specific glycoprotein. This refers to a known protein also known as B29, Ig-β, or AGM6. The protoreceptor is a multimeric complex containing antigen-specific components, surface immunoglobulin (Ig). CD79b is a B cell antigen component of Ig- It is a β protein. All CD79b isoforms and variants are "CD79b It is included in ". The amino acid sequences of various isoforms are GenBank accessions Numbers AAH32651.1, EAW94232.1, AAH02975.2, NP_0 Available from 00617.1 and NP_001035022.1. Full length CD7 The amino acid sequence of sequence 9b is shown below. The sequence includes the extracellular domain (residues 29-159) and It contains cytoplasmic domains (residues 181-229). MARLALSPVPSHWMVALLLLLSAEPVAARSEDRYRNPK GSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWK QEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGI YFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKD GIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGL DIDQTATYEDIVTLRTGEVKWSVGEHPGQE (Sequence ID 282)

[0100] Chimeric antigen receptor The present invention generally involves genetically modifying a gene to stably express a desired chimeric antigen receptor. Regarding the use of T cells. Chimeric antigen receptors (CARs) are T cell signaling domains. An artificially constructed hybrid protein containing the antigen-binding domain of an antibody linked to it. It is a protein or polypeptide (scFv). The characteristics of CARs include non-MHC restricted selection. Redirecting T cell specificity and reactivity toward the selected target, monoclonal antibody One notable feature is its ability to utilize its antigen-binding properties. Through recognition of non-MHC restriction antigens, C T cells that express AR can recognize antigens independently of antigen processing. Therefore, it bypasses the main mechanism of tumor escape. Furthermore, when expressed in T cells, C AR is favored by endogenous T cell receptor (TCR) alpha and beta chains. It does not dimerize.

[0101] The CAR described herein comprises at least an extracellular antigen-binding domain and a transmembrane domain. intracellular signals containing functional signaling domains derived from the following stimulating molecules Includes a signaling domain (also referred to herein as the "cytoplasmic signaling domain") , provides recombinant polypeptide constructs. T cells expressing CAR are specified herein. Therefore, these are called CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms These terms are used interchangeably in this specification. Cells secure the antibody-binding domain on their surface. It can be genetically modified to be expressed consistently, and novel MHC-independent antigen specificity can be achieved. Grant.

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

[0103] When the term "intracellular signaling domain" is used herein, it refers to a molecule. This refers to the intracellular portion of a protein. This is the functional part of the protein, acting as a second messenger. Cells regulate cellular activity through signaling pathways defined by their generation. It operates by transmitting information internally, or by responding to such messengers. It acts by functioning as an effector. The intracellular signaling domain is C It generates signals that promote the immune effector function of AR-containing cells (e.g., CAR-T cells). To accomplish. For example, an example of immune effector function in CAR-T cells is cytolytic activity. Other examples include helper activity (including cytokine secretion).

[0104] In one embodiment, the intracellular signaling domain is the primary intracellular signaling domain. It may include. Examples of primary intracellular signaling domains include primary stimulus or antigen-dependent blemishes. Examples include those derived from molecules involved in sigma. In one embodiment, intracellular sigma The signaling domain may include a co-stimulatory intracellular domain. Examples of "ins" include those derived from molecules involved in co-stimulatory signals or antigen-independent stimuli. For example, in the case of CAR-T cells, the primary intracellular signaling domain is located in the T cell. The receptor may contain a cytoplasmic sequence, and the co-stimulatory intracellular signaling domain may be a co-receptor or co-stimulatory domain. It may contain cytoplasmic sequences derived from highly active molecules.

[0105] The primary intracellular signaling domain is an immune receptor tyrosine-based activation motif or It may contain a signaling motif known as ITAM. Primary cytoplasmic signaling sequence Examples of ITAMs containing CD3-ζ, FcRγ, FcRβ, CD3γ, CD3 δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP Examples include, but are not limited to, those derived from 10 and DAP12.

[0106] The terms "ζ" or alternatively "ζ chain", "CD3-ζ", or "TCR-ζ" refer to Ge The protein provided as nBank accession number BAG36664.1, or From non-human species, such as rats, rabbits, primates, mice, rodents, monkeys, and apes. Defined as an equivalent residue, it is called the "ζ-stimulating domain" or, alternatively, the "CD3-ζ-stimulating domain". The "TCR-ζ stimulating domain" or "TCR-ζ stimulating domain" functionally provides the initial signals necessary for T cell activation. It is defined as an amino acid residue from the cytoplasmic domain of the ζ chain sufficient for transmission. In this case, the cytoplasmic domain of ζ is GenBank accession number BAG36664. Residues 52-164 of compound 1, or their functional orthologues, in non-human species, e.g., mouse. It contains equivalent residues derived from rodents, monkeys, apes, etc. In one embodiment, it is called "ζ-stimulated domain "n" or "CD3-ζ stimulating domain" is the sequence provided as Sequence ID No. 28, or the sequence Number 41 and at least 50, at least 55, at least 60, at least 65, less 70, at least 75, at least 80, at least 85, at least 90, less 95, at least 96, at least 97, at least 98, or at least 99 These are sequences that have % sequence identity.

[0107] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby affecting T cells. Co-stimulatory responses (e.g., proliferation, but not limited to) mediated by allogeneic tumors on T cells This refers to a co-stimulatory partner. A co-stimulatory molecule is an antigen receptor or its ligament necessary for an efficient immune response. These are cell surface molecules other than brands. Co-stimulatory molecules include 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) These are some examples, but are not limited to them.

[0108] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. The children belong to the following protein families: TNF receptor proteins, immunoglobulin-like proteins. Calcium, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM) It can be represented as a protein, and as an activated NK cell receptor. Examples of such molecules include For example, 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 Examples include ligands that specifically bind to CD160, B7-H3, and CD83. .

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

[0110] The term "4-1BB" or alternatively "CD137" refers to GenBank accession The amino acid sequence provided as number AAA62478.2, or for non-human species, e.g., Mau The TNFR superfamily has equivalent residues from rodents, monkeys, apes, and other animals. The "4-1BB Co-Stimulation Domain" refers to the members of the GenBank accession number. Amino acid residues 214-255 of AAA62478.2, or non-human species, e.g., mouse It is defined as equivalent residues from rodents, monkeys, and apes. In one embodiment, The "4-1BB co-stimulatory domain" or "CD137 co-stimulatory domain" is represented as Sequence ID No. 27. The provided sequence, or from non-human species such as mice, rodents, monkeys, apes, etc. Valuable residues, or at least 50, at least 55, at least 60 of SEQ ID NO: 40 At least 65, at least 70, at least 75, at least 80, at least 85, less at least 90, at least 95, at least 96, at least 97, at least 98, if k is a sequence that has at least 99% sequence identity.

[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 is selected or modified by amino acid substitution, and To avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, Interaction with other members of the receptor complex can be minimized. One example of implementation. In this state, the transmembrane domain includes the CD8α hinge domain.

[0112] In some embodiments, the cytoplasmic signaling domain is less than the definitions provided herein. It further includes one or more functional signaling domains derived from at least one co-stimulatory molecule. In one embodiment, the co-stimulatory molecules are 4-1BB (i.e., CD137), CD27, CD Selected from 3-ζ and / or CD28. CD28 is important in T cell costimulation. It is a T cell marker. CD27 is a member of the tumor necrosis factor receptor superfamily. It functions as a co-stimulating immune checkpoint molecule. 4-1BB is potent on T cells. It transmits co-stimulatory signals, promotes differentiation, and enhances the long-term survival of T lymphocytes. CD3- ζ associates with the TCR to generate a signal and activates an immune receptor tyrosine-based motif. It contains (immunoreceptor tyrosine-based activation motifs, ITAM). In the application mode, the co-stimulatory molecule is MyD88 or CD40.

[0113] In one embodiment, the CAR comprises an intracellular hinge domain containing CD8 and CD28, 4-1 It includes BB and an intracellular T cell receptor signaling domain containing CD3-ζ. In this embodiment, the CAR comprises an intracellular hinge domain and CD28, 4-1BB, and CD3 -The domain comprises an intracellular T cell receptor signaling domain containing ζ, and the hinge domain is All or part of the extracellular region of CD8, CD4, or CD28, or all or part of the antibody constant region. Parts, FcRIIIA receptor, IgG hinge, IgM hinge, IgA hinge, IgD hinge , IgE hinge, or all or part of Ig hinge. IgG hinge includes IgG1, I gG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, Ig E may be derived from a chimera thereof.

[0114] The CAR described herein comprises at least an extracellular antigen-binding domain and a transmembrane domain. For example, intracellular signals containing functional signaling domains derived from stimulating molecules defined below The signaling domain (also referred to herein as the "cytoplasmic signaling domain") and The present invention provides recombinant polypeptide constructs containing the following:

[0115] In one embodiment, the CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and a stimulating molecule. An intracellular signaling domain containing a functional signaling domain derived from and containing It contains a MELLA fusion protein. In one embodiment, the CAR has an extracellular antigen recognition domain and a membrane The transconducting domain, the functional signaling domain derived from the co-stimulator molecule, and the domain derived from the stimulator molecule. An intracellular signaling domain containing a functional signaling domain, and a chimeric fusion domain containing It contains a combined protein. In one embodiment, the CAR has an extracellular antigen recognition domain and a transmembrane domain. Main and at least two functional signaling domains derived from one or more co-stimulatory molecules Intracellular signaling domains containing functional signaling domains derived from ions and stimulating molecules It contains a chimeric fusion protein that includes n and .

[0116] The CAR of the present invention has a CD28 and / or 4-1BB signaling domain. The body may contain, or any other desired cytoplasmic sulfate useful in the context of the CAR of the present invention It can be designed to be used in combination with . The signaling domain of CAR may further include the CD3-ζ signaling domain. For example, the cytoplasmic domain of CAR The main components are the CD3-ζ, 4-1BB, and CD28 signaling modules, as well as These combinations are possible, but not limited to, these. Therefore, the present invention This provides CAR T cells and methods for their use in adoptive therapy.

[0117] This disclosure relates to the CARs, nucleic acids, polypeptides, and proteins described herein. It further provides variants (e.g., functional variants). A "variant" is, for example, a replacement or insertion. or one or more modifications, such as deletion, to the reference polypeptide or reference polynucleotide Refers to a polypeptide or polynucleotide different from the above. When used herein, "functional The term "variant" refers to a variant that is substantially or significantly different from the parent CAR, polypeptide, or protein. This refers to CARs, polypeptides, or proteins that have sequence identity or similarity, and these A functional variant is a variant of a CAR, polypeptide, or protein. It retains activity. Functional variants include, for example, the CARs and polypeptides described herein. , or encompassing variants of proteins (parent CAR, polypeptide, or protein), Labeled to the same degree, the same degree, or a higher degree as the parent CAR, polypeptide, or protein. It retains the ability to recognize target cells. With respect to parent CARs, polypeptides, or proteins, Functional variants are, for example, amides of parent CAR, polypeptide, or protein. The no-acid sequence accounts for at least approximately 30%, 40%, 50%, 60%, 75%, and 80% of the total. Approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, They may be approximately 97%, 98%, 99%, or more identical.

[0118] A functional variant is, for example, a parent CAR having at least one conserved amino acid substitution. , may include a polypeptide or protein amino acid sequence. In another embodiment, functional The variant is a parent CAR, polypeptide having at least one non-conservative amino acid substitution. , or may include the amino acid sequence of a protein. In this case, non-conservative amino acid substitutions are functional. It is not possible to suppress or inhibit the biological activity of the target variant. This allows for the improvement of the biological activity of the functional variant, and as a result, the functional variant The biological activity of the parent CAR, polypeptide, or protein is increased compared to the parent CAR.

[0119] The amino acid substitutions of CAR in the present invention may be conservative amino acid substitutions. The exchange is known in the art and has specific physical and / or chemical properties. Replace one amino acid with another amino acid that has the same or similar chemical or physical properties. This includes amino acid substitutions. For example, a conservative amino acid substitution is an acid that is substituted with another acidic amino acid. A sexual amino acid (e.g., Asp or Glu), substituted with another amino acid having a nonpolar side chain. Amino acids having nonpolar side chains (e.g., Ala, Gly, Val, Ile, Leu, M) (e.g., et, Phe, Pro, Trp, Val), basic amino acids substituted with other basic amino acids Amino acids (Lys, Arg, etc.), polar side chains substituted with other amino acids that have polar side chains These may be amino acids that contain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.) ru.

[0120] CARs, polypeptides, or proteins are defined by a specific amino acid sequence or as described herein. Essentially, the sequence can become, and as a result, other components, such as other amino acids, can become functional. It does not substantially alter the biological activity of riantoxin.

[0121] CARs, polypeptides, and proteins (functional portions and functional parts) of embodiments of the present disclosure (including riant) can be of any length, that is, it can contain any number of amino acids, However, CAR, polypeptide, or protein (or functional part or its functional variant) Ants exhibit biological activity, such as the ability to specifically bind to antigens and disease cells within the host. The ability to detect cells (e.g., cancer cells), or the ability to treat or prevent disease within a host. It holds such things. For example, polypeptides have a length of about 50 to about 5000 amino acids, for example Approximately 50, 70, 75, 100, 125, 150, 175, 200, 2 25, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 4 25, approximately 450, approximately 475, approximately 500, approximately 525, approximately 550, approximately 575, approximately 600, approximately 6 25, approximately 650, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, approximately 800, approximately 8 25, approximately 850, approximately 875, approximately 900, approximately 925, approximately 950, approximately 975, approximately 1000 or The amino acid length may be even longer. The polypeptide of the present invention also includes oligopeptides. .

[0122] CAR, polypeptide, and protein according to embodiments of the present invention (functional portion and (Including functional variants) are synthetic amino acids instead of one or more naturally occurring amino acids. It may contain an acid. Such synthetic amino acids are known in the art, for example, For example, aminocyclohexanecarboxylic acid, norleucine, α-aminon-decanoic acid, ho Moserine, 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-diaminobutyric acid Minopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carbboxy Phenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenyl Lysine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N '-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydrox Cyridine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexa α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, amino Examples include monoamide ronate and α-tert-butylglycine.

[0123] CAR, polypeptide, and protein (functional portion and functional portion) according to embodiments of the present invention (including riantos) can undergo post-translational modifications. These include glycosylation, esters. Telation, N-acylation, amidation, carboxylation, phosphorylation, esterification, for example, di It can be cyclized via sulfide bridges, or converted into an acid addition salt. In some embodiments, these are dimerized, polymerized, or conjugated. It is being done.

[0124] CARs, polypeptides, and / or proteins (functional portions and so) according to embodiments of the present invention (including functional variants) can be obtained by methods known in the art. A suitable method for the de novo synthesis of polypeptides and proteins is described by Chan et al. l.,Fmoc Solid Phase Peptide Synthesis,Ox Ford University Press,Oxford,United King dom,2000, Peptide and Protein Drug Analyses is, ed. Reid, R., arcel Dekker, Inc., 2000, and E. Pitope Mapping, ed. Westwood et al., Oxford University Press,Oxford,United Kingdom, References such as 2001 are cited. Furthermore, polypeptides and proteins are standard. Using the nucleic acids described herein, a recombinant recombination method may be produced. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed.,Cold Spring H arbor Press, Cold Spring Harbor, NY2001, and Ausubel et al.,Current Protocols in Mo lecular Biology,Greene Publishing Associ See ates and John Wiley & Sons, NY, 1994. Furthermore, the CAR, polypeptide, and protein (functional portion and its functional portion) of the present invention Some of the variants (including) are isolated from sources such as plants, bacteria, insects, and mammals and / Alternatively, it can be purified. Methods for isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and / or proteins (functional portions) described herein. (and its functional variants) can be commercially synthesized. CARs, polypeptides, and proteins are synthesized, recombinant, isolated, and / or purified. It is possible.

[0125] To generate recombinant nucleic acids used to produce the polypeptides described herein Examples of modified nucleotides that can be used include 5-fluorouracil, 5-br Romouracil, 5-Chloruracil, 5-Iodouracil, Hypoxanthine, Xanthine , 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethyl Thiaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, di Hydrouracil, N 6 - Substitute adenine, 7-methylguanine, 5-methylaminomethyl Lacil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqu Osin, 5"-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methyl Thio-N 6 -Isopentenyl adenine, uracil-5-oxyacetic acid (v), weybutoxo Syn, Pseudouracil, Quosin, Beta-D-GalactosylQuosin, Ino Shin, N 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine , 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thio Lacil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester Tel, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-di Examples include, but are not limited to, aminopurines.

[0126] Nucleic acids are CARs, polypeptides, proteins, or functional parts or their functions. Includes any isolated or purified nucleotide sequence encoding any of the variants It can be seen. Alternatively, a nucleotide sequence is a degenerate nucleotide sequence of any of the sequences. Alternatively, it may include combinations of degenerate arrangements.

[0127] Some embodiments of the present invention also provide isolated or purified nucleic acids, wherein the nucleic acids are Nucleotide sequences that are complementary to any of the nucleotide sequences of the nucleic acids described in the specification. or stringent elements in any nucleotide sequence of the nucleic acids described herein The item contains a nucleotide sequence that hybridizes below.

[0128] Nucleotide sequences that hybridize under stringent conditions are highly stringent. Hybridization is possible under stringency conditions. "High stringency conditions" refers to nucleotides. The sequence is detected in a stronger amount than nonspecific hybridization in the target sequence (as specified herein). To specifically hybridize to any of the nucleotide sequences of the nucleic acids listed in [the relevant section]. This means that high stringency conditions are polynucleotides with precisely complementary sequences. Alternatively, a polynucleotide containing only a few scattered mismatches, and a nucleotide sequence and Random distribution resulting in several matched small regions (e.g., 3-12 bases) Includes conditions for identifying the column. Such complementary subregions are complete with 14-17 or more bases. It melts more easily than the full-length complement and undergoes high-stringency hybridization. This makes them easily identifiable. Relatively high stringency conditions are, for example, about It is provided by approximately 0.02 to 0.1 M NaCl or equivalent at a temperature of 50 to 70°C. This includes conditions such as low salinity and / or high temperature. Such high stringency conditions are nuclear Even if there is a mismatch between the rheotide sequence and the template or target strand, it is mostly Not permitted and particularly suitable for detecting the expression of any of the CARs described herein. The condition is that it can become more stringent by gradually increasing the amount of formamide added. However, this is generally understood.

[0129] In one embodiment, the nucleic acid of the present invention can be incorporated into a recombinant expression vector. The diagram shows a recombinant expression vector comprising any of the nucleic acids of the present invention. When referring to a "recombinant expression vector," the term is used to describe mRNA and protein expressed by host cells. Genetically modified oligonucleotides that enable the expression of polypeptides or peptides. This refers to a nucleotide or polynucleotide construct, in which case the construct is mR. It contains nucleotide sequences encoding NA, proteins, polypeptides, or peptides, Vectors express mRNA, proteins, polypeptides, or peptides within cells. Under conditions sufficient for this, the vectors are brought into contact with the cells. The vectors described herein, as a whole It does not exist in nature. However, some vectors may exist in nature. Recombinant expression vectors are DNA and RNA (which can be single-stranded or double-stranded, and can be synthesized). Or may be obtained in part from natural sources and may contain natural, non-natural or modified nucleotides. Recombination may include, but is not limited to, any type of nucleotide. The expression vector is either a naturally occurring nucleotide linkage or a nucleotide linkage that does not exist in nature. This may include rheotide linkages, or both types of linkages. These may not exist naturally or may be modified. Nucleotides or internucleotide links do not inhibit the transcription or replication of the vector.

[0130] In one embodiment, the recombinant expression vector of the present invention can be used with any suitable recombinant expression vector. It may be used to transform or transfect any suitable host. Suitable vectors include plasmids and viruses, which are used for reproduction and proliferation. Examples include vectors designed for expression or both. These vectors are pUC. Series (Fermentas Life Sciences, Glen Burnie) ,Md.), pBluescript series (Stratagene, LaJolla , Calif.), pET series (Novagen, Madison, Wis.), p GEX series (Pharmacia Biotech, Uppsala, Sweden) ), and from the pEX series (Clontech, Palo Alto, Calif.) The following groups can be selected: λGT10, λGT11, λEMBL4, and λNM1149. Using bacteriophage vectors such as λZapII (Stratagene) This is possible. Examples of plant expression vectors include pBI01, pBI01.2, and pBI1 Examples include 21, pBI101.3, and pBIN19 (Clontech). Animal development Examples of current vectors include pEUK-Cl, pMAM, and pMAMneo(Clont Examples include (ech). Recombinant expression vectors are viral vectors, for example, Retrovir This could be a ruth vector, such as a gamma retrovirus vector.

[0131] In one embodiment, the recombinant expression vector of the present invention is, for example, the Sambrook vector described above. Standard recombinants described in et al. (above) and Ausubel et al. (above) The expression vector construct is prepared using DNA technology, and is circular or linear. It can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The manufacturing process involves, for example, ColE1, SV40, 2μ plasmid, λ, and bovine papillomavirus. It can be derived from things like that.

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

[0133] Recombinant expression vectors allow for the selection of transformed or transfected hosts. It may contain one or more marker genes. The marker genes may indicate biocide resistance (e.g., Resistance to antibiotics, heavy metals, etc., nutritional requirements, providing protonutrient supply in the host This includes complementary genes, etc. Suitable marker genes for the expression vectors described include, for example, Ne Omycin / G418 resistance gene, histidinol x resistance gene, histidinol resistance gene Examples include genes, tetracycline resistance genes, and ampicillin resistance genes.

[0134] Recombinant expression vectors are CARs, polypeptides, or proteins (functional portions and The nucleotide sequence encoding the functional variant (including that functional variant), or CAR, polyp Complementary to or related to the nucleotide sequence encoding a nucleotide or protein. Nucleotide sequences that hybridize to natural or normative promotional materials, operably linked. It may include promoters. Promoter selection (e.g., strong, weak, tissue-specific, inducible, and (and development-specific) is within the scope of the art of those skilled in the art. Similarly, promoting nucleotide sequences Combining it with other methods is also within the scope of the skills of those skilled in the art. The promoter is a non-viral promoter. Motor or virus promoter, for example, cytomegalovirus , CMV promoter, RSV promoter, SV40 promoter, or mouse stem cell It could be a promoter found in the long terminal repeats of vesicular viruses.

[0135] Recombinant expression vectors are CARs, polypeptides, or proteins (functional portions and The nucleotide sequence encoding the functional variant (including that functional variant), or CAR, polyp Complementary to or related to the nucleotide sequence encoding a nucleotide or protein. Enhancer elements are manipulably ligated to nucleotide sequences that hybridize to a hybridized nucleotide sequence. One or more additional modulatory elements such as the 5' and 3' UTR, or terminator array It can include [something].

[0136] Recombinant expression vectors are used for either transient expression or stable expression, or either of the following: It can be designed for both. Recombinant expression vectors can also be used for constitutive or inducible expression. It can be made for that purpose.

[0137] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. At that time, the term "suicide gene" refers to a gene that causes cells that express a suicide gene to die. It refers to a gene. A suicide gene is a gene that causes cells expressing the gene to react to drugs (e.g., drugs). To confer sensitivity, preventing cell death when cells come into contact with or are exposed to the drug. It may be a gene that causes suicide. Suicide genes are known in the field of technology, for example, Pure herpesvirus (HSV) thymidine kinase (TK) gene, cytosine deaminase Examples include purine nucleoside phosphorylase and nitroreductase.

[0138] CAR, polypeptide, or protein (either its functional part or variant) (including), host cells, nucleic acids, recombinant expression vectors, host cell populations, or antibodies, A conjugate containing any of these antigen-binding sites, for example, a bioconjugate, This falls within the scope of the present invention. Conjugates and methods for synthesizing conjugates are generally, Known in the relevant technical field (e.g., Hudecz, F., Methods Mo l.Biol.298:209-223(2005) and Kirin et al.,I See norg Chem. 44(15):5405-5415 (2005).

[0139] One embodiment of the present invention involves binding to the epitope of the CAR of the present invention, for example, specifically binding The present invention further provides an antibody or its antigen-binding portion.

[0140] The antibody may be any type of immunoglobulin known in the art. Globulins are classified into five main classes: IgA, IgD, IgE, IgG, and Ig M can be assigned to IgA and IgG, which are isotypes IgA1, IgA2, and IgG. 1. Further subdivided into IgG2, IgG3, and IgG4. The antibody light chains of vertebrate species are: Based on the amino acid sequences of those constant domains, two types are identified: kappa (κ) and lambda (λ). It can be assigned to one of the types. The antibody is of any class or isotype. obtain.

[0141] Antibodies include monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies. Body, polyclonal, antigen-binding fragment, bispecific or multispecific antibody, monomer, dimer, Tetramer or multimer antibodies, single-chain antibodies, domain antibodies, and antigenic antibodies of the required specificity. Immunoglobulin molecules containing any other modified stereochemistry including the fusion site, It contains molecules. Antibodies are naturally occurring antibodies, for example, mammalian antibodies (e.g., mice, primates). Isolation and / or from animals (such as mice, rabbits, goats, horses, chickens, hamsters, and humans) This could be a purified antibody. Alternatively, the antibody may be genetically engineered (e.g., genetically engineered). It could be a manipulated antibody.

[0142] Humanized antibodies have antigen-binding sites derived from non-human species, and the variable region framework is, Derived from human immunoglobulin sequences. Human antibodies possess both a framework and an antigen-binding site. The latter has heavy chain variable regions and light chain variable regions derived from human-derived sequences.

[0143] Furthermore, antibodies possess any level of affinity or binding activity to the functional portion of CAR. Obtain. In some embodiments, the antibody has an affinity (K) within a certain range. D ) and the CD79b antigen They can be combined. In one embodiment of the present invention, and in the numbered embodiments listed below, In each of our embodiments and in some of the embodiments of all of them, those skilled in the art have put into practice It is determined by the surface plasmon resonance or coupled equilibrium exclusion (Kinexa) method applied. Sea urchins and antibodies have high affinity, for example, about 10 -7 For KD below M, for example, limited to Shina However, 1 to 9.9 (or 1, 2, 3, 4, 5, 6, 7, 8, or 9, etc., within this range) (Any range or value) × 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M, or any range that falls within it It binds to the CD79b antigen at a KD level or value. An example of affinity is 1 × 10⁻¹⁰. -8 More than M Below. Another example of affinity is 1 × 10 -9 It is M or less.

[0144] A method for testing an antibody's ability to bind to any functional part of a CAR is described in the technical section. Known in the field, and applicable to any antibody-antigen binding assay, e.g., radioimmunoassay. (radioimmunoassay, RIA), Western blot, enzyme-coupled immunosorbent assay (enzyme) ME-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays, etc. These are some examples.

[0145] Suitable methods for producing antibodies are known in the art. For example, standard methods The ibridomas method is, for example, described by Kohler and Milstein, Eur.JI. mmunol.,5,511519(1976),Harlow and Lane(e ds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), I mmunobiology,5th Ed.,Garland Publishing, It is described in New York, NY (2001). Alternatively, other methods, examples For example, the EBV-hybridoma method (Haskard and Archer, J.Imm). unol.Methods,74(2),36167(1984),and Roder et al.,Methods Enzymol.,121,14067(1986) ), and bacteriophage vector expression systems (e.g., Huse et al., Sci See ence, 246, 1275-81 (1989) etc. in the relevant technical field. This is known in [location]. Furthermore, methods for producing antibodies in non-human animals are, for example, [see US patent]. Nos. 5,545,806, 5,569,825, and 5,714,352, This is also described in U.S. Patent Application Publication No. 2002 / 0197266(A1).

[0146] It is also possible to generate antibodies using phage display. In this regard, antibodies Phage libraries encoding antigen-binding variable (V) domains are standard molecular biological It can be produced using scientific and recombinant DNA technology (e.g., Sambrook See et al. (above) and Ausubel et al. (above). A phage encoding a variable region with the desired specificity will then transmit the desired antigen (i.e., C For specific binding to D79b), selected, including the selected variable domain, either whole or partially. A fractional antibody is reconstituted. The nucleic acid sequence encoding the reconstituted antibody is a hybridoma. It is introduced into suitable cell lines such as myeloma cells used for production, and as a result, monoclonal Antibodies possessing antibody properties are secreted by cells (for example, Janeway et al. l. (above), Huse et al. (above), and U.S. Patent No. 6,265,150 (See reference).

[0147] Antibodies are transgenic of specific heavy and light chain immunoglobulin genes. It can be produced by a sgenic mouse. Such a method is already in use in the art. This is knowledge, for example, U.S. Patent No. 5,545,806 and No. 5,569,825, and Janeway et al., as listed above.

[0148] Methods for producing humanized antibodies are known in the art, for example, Janew ay et al., U.S. Patent No. 5,225,539, No. 5,585,089, and No. Patent No. 5,693,761, European Patent No. 0239400(B1), and British Patent No. 218 It is described in U.S. Patent No. 8638. Humanized antibodies are also described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 It can also be produced using the antibody resurfacing technique described in (1994).

[0149] When used herein, antibodies may be multiple or single chains, or intact immunoglobulins. The antibody may be immunoglobulin, and may be derived from natural or recombinant sources. It can be a tetramer of n molecules.

[0150] In some embodiments, the antibody is a bispecific antibody. VL and / or young of existing antibodies or VH area, or newly identified VL and VH areas as described herein. The region may be genetically engineered to produce a bispecific full-length antibody. The body uses techniques such as those described below to form bispecific antibodies against It can be produced by modulating CH3 interactions in the Fc: US 7,695 Patent No. 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 / 028637 Patent No. 4, U.S. Patent Publication No. 2011 / 0123532, International Patent Publication No. 2011 / 131746 International Publication No. 2011 / 143545, or U.S. Patent Publication No. 2012 / 014987 These include those described in publication No. 6. For example, the bispecific antibody of the present invention is described in International Publication No. According to the method described in issue 2011 / 131746, two types of single-cell cultures were cultured in a cell-free environment. An asymmetric mutation is introduced into the CH3 region of an isomer homodimer antibody, altering the disulfide bond. Under reducing conditions that induce sexualization, two types of parental monospecific homodimer antibodies produce bispecific antibodies. It may be produced by forming a telodimer antibody. In this method, the first single The specific bivalent antibody and the second monospecific bivalent antibody promote the stability of the heterodimer. These antibodies are genetically engineered to have a specific substitution in three domains, but these antibodies , cysteine ​​in the hinge region is sufficient reducing element to isomerize the disulfide bond. Under these circumstances, they are incubated together, and as a result, the Fab arm can be replaced to create a dual special Heterozygous antibodies are produced. The incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents that can be used include 2-mercaptoethylamine (2-MEA) and dithioslate. Dithioerythritol (DTT), dithioerythritol (DTE), glutathione, tris(2-carb) Xyethyl(Phosphine) (TCEP), L-cysteine, and beta-mercaptoethano The compounds are preferably 2-mercaptoethylamine, dithiothreitol, and triglycerides. A reducing agent selected from the group consisting of (2-carboxyethyl)phosphine. at a temperature of at least 20°C, in the presence or absence of at least 25 mM 2-MEA At least 0.5 mM dithiothreitol, pH 5-8, for example, pH 7.0 or An incubation period of at least 90 minutes at pH 7.4 may be used.

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

[0152] The term "scFv" refers to an antibody fragment containing at least one variable region of the light chain and a heavy chain. This refers to a protein containing at least one antibody fragment that includes a variable region of . In the application configuration, the light chain variable region and the heavy chain variable region are connected via short, flexible polypeptide linkers. They are linked together in a continuous chain and can be expressed as a single-chain polypeptide, and scFv is its original The specificity of the intact antibody is preserved. Unless otherwise specified, when used herein, scFv may have the VL and VH variable regions in any order, for example, polyp Regarding the N-terminus and C-terminus of the cyd, scFv may include VL-linker-VH. It may also contain VH-linker-VL.

[0153] The VH and VL domains identified herein should be incorporated into the scFv format. The resulting scFv can be bonded to CD79b and its thermal stability can be determined using known methods. It can be evaluated. The combination is ProteOn XPR36, Biacore3000. Alternatively, using a KinExA instrument, ELISA, or a competitive binding assay known to those skilled in the art. It can be evaluated using purified scFv or E. coli supernatant or expressed Evaluation can be performed using lysed cells containing scFv. Test scFv CD79 The affinity for b was measured under different conditions (e.g., molar osmotic pressure, pH). This may differ depending on the case. Therefore, affinity and other binding parameters (e.g., K D , K on , K off The measurement of ) is usually performed under standard conditions and using standardized buffer. Thermal stability is measured at high temperatures such as 50°C, 55°C, or 60°C for 5 minutes, 10 minutes, and 1 minute. Heat the test scFv for a period of time such as 5 minutes, 20 minutes, 25 minutes, or 30 minutes, and the test scF This can be evaluated by measuring the binding of v to CD79b. Unheated scF When compared to the v sample, scFv, which retains a bond equivalent to that of CD79b, has better thermal stability. It is said to be.

[0154] The cD79b antigen-binding domain exhibits improved thermal stability compared to the parent antibody SN8. The shown may include sequences encoding mutants. Genetic engineering that can confer improved thermal stability. The positions where this occurs are residues M12, I20, R40, and A79 in VH (SEQ ID NO: 283). Residue numbering according to SN8_VH, as well as L4, D32, F51, V82 in VL, and includes A87 (residue numbering according to SN8_VL of SEQ ID NO: 284).

[0155] In recombinant expression systems, the linker is a peptide linker, and any naturally occurring amino acid It may contain acids. Exemplary amino acids that may be present in the linker are Gly, Ser Pro, and T These are hr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker is , precise higher-order structures relative to each other, so as to retain desired activity such as binding to CD79b It is necessary to have an appropriate length to connect VH and VL in a manner that forms a [specific configuration]. ru.

[0156] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker - has an amino acid length of approximately 10 to 40. In some embodiments, the linker is approximately 1 The amino acid length is 0 to 35. In some embodiments, the linker is approximately 10 to 30. This is the length of the amino acids. In some embodiments, the linker is about 10 to 25 amino acids. It is long. In some embodiments, the linker is about 10 to 20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker has a length of 6 amino acids. It has a length of 7 amino acids. In some embodiments, the linker has a length of 8 amino acids. In some embodiments, the linker is 9 amino acids long. In the application form, the linker has a length of 10 amino acids. In some embodiments, the linker - is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. It is the length of the amino acid. In some embodiments, the linker is the length of 13 amino acids. In some embodiments, the linker has a length of 14 amino acids. In some embodiments, The linker has a length of 15 amino acids. In some embodiments, the linker has a length of 16 amino acids. The length is 17 amino acids. In some embodiments, the linker has a length of 17 amino acids. In some embodiments, the linker has an amino acid length of 18. Morphologically, the linker has a length of 19 amino acids. In some embodiments, the linker It has a length of 20 amino acids. In some embodiments, the linker has a length of 21 amino acids. It is acidic. In some embodiments, the linker is 22 amino acids long. In one embodiment, the linker has a length of 23 amino acids. In some embodiments, The linker has a length of 24 amino acids. In some embodiments, the linker has 25 amino acids. This is the amino acid length. In some embodiments, the linker has a length of 26 amino acids. In some embodiments, the linker has a length of 27 amino acids. In this configuration, the linker has a length of 28 amino acids. In some embodiments, the linker is It has a length of 29 amino acids. In some embodiments, the linker has a length of 30 amino acids. In some embodiments, the linker has a length of 31 amino acids. In some embodiments, the linker has a length of 32 amino acids. In some embodiments, phosphorus The linker has a length of 33 amino acids. In some embodiments, the linker has a length of 34 amino acids. It is the amino acid length. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker has a length of 36 amino acids. The linker has a length of 37 amino acids. In some embodiments, the linker is 3 It has an amino acid length of 8. In some embodiments, the linker has an amino acid length of 39. Yes, in some embodiments, the linker is 40 amino acids long. Examples of linkers include glycerin-rich linkers, glycerin and ser-containing linkers, and glycerin and ser-containing linkers. Ala-containing linkers, Ala and Ser-containing linkers, and other flexible linkers ru.

[0157] Other linker sequences are immunoglobulin heavy chain or light chain isotypes. The link hinge region may include a portion of CL or CH1. An example linker that may be used is: This is shown in Table 1. Additional linkers are described, for example, in International Publication No. 2019 / 060695. It is being done.

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

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

[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 includes the amino acid sequence of SEQ ID NO: 58.

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

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

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

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

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

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

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

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

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

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

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

[0188] In some embodiments, the linker includes 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 portion consists of Fab, F(ab')2, dsFv, sFv, diabody, and It may be any part having at least one antigen-binding site, such as a triabody.

[0195] In some embodiments, the antigen-binding fragment is a heavy chain complementarity determining region. n complementarity determining regions, HCDR)1, 2 and / or 3, light chain Light chain complementarity determining regions (LCDR) 1, 2, and / or 3, heavy chain variable region (VH), or light chain variable region (VL), Fab, F (ab')2, Fd, and Fv fragments, as well as one VH domain or one VL A domain antibody (dAb) that contains (for example, consists of) one of the domains. The VH domain and VL domain are linked together via a linker, such as a synthetic linker. It is possible.

[0196] "Complementarity determining regions (CDRs)" are found in antibodies. It is an antigen-binding site. CDR can be defined using various terms: (i) within VH There are three in (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCD The complementarity-determining region (CDR) of R2 (LCDR3) is based on sequence diversity (Wu and Kabat,J Exp Med 132:211-50,1970;Kaba tet al.,Sequences of Proteins of Immunol ogical Interest,5th Ed.Public Health Ser vice,National Institutes of Health,Bethe (sda, Md., 1991). (ii) Three within VH (H1, H2, H3) and within VL The three (L1, L2, L3) "highly variable regions," "HVR," or "HV" are Chot hia and Lesk (Chothia and Lesk Mol Biol 196: As defined by 901-17, 1987, antibodies are highly variable in structure. This refers to the area of ​​the variable domain. International ImMunoGeneTic The s(IMGT) database (http: / / imgt_org) is a database of antigen-binding sites. Provides quasi-numbering and definitions. Correspondence between CDR, HV, and IMGT summary descriptions is provided by Le franc et al., Dev Comparat Immunol 27:55- It is described in 77, 2003. The terms "CDR" and "HCDR1" used herein are used in this specification. "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCD The term "R3" refers to the above-mentioned Kabat, Cho, unless otherwise specified herein. CDR as defined by either thia or the method described by IMGT include.

[0197] Furthermore, the antibody or its antigen-binding portion may be, for example, a radioactive isotope, a fluorophore (for example) Fluorescein isothiocyanate (FITC), Coerythrin (PE), enzymes (e.g., alkaline phosphatase, West A detectable label such as horseradish peroxidase and elemental particles (e.g., gold particles) It can be modified to include.

[0198] Examples of detectable labels include radioactive isotopes, magnetic beads, metallic beads, and colloids. Particles, fluorescent dyes, electron density reagents, enzymes (for example, commonly used in ELISA) ), biotin, digoxigenin, hapten, luminescent molecule, chemiluminescent molecule, fluorescent dye, fluor Lophore, fluorescent quencher, colored molecule, radioisotope, scintillates, avi Zin, streptavidin, protein A, protein G, antibody or fragment thereof, polyhysteresis Thidine, Ni2+, Flag tag, myc tag, heavy metal, enzyme, alkaline phosphatase Peroxidase, luciferase, electron donor / receptor, acridinium ester, Examples include colorimetric substrates.

[0199] Detectable labels spontaneously, for example, when the detectable label is a radioactive isotope. It may emit a signal. In other cases, the detectable marker is a result of stimulation by an external field. And it emits a signal. Suitable dyes include, for example, 5(6)-carboxyfluorescein. IRDye 680RD maleimide, or IRDye 800CW ruthenium Any commercially available dyes such as lipyridyl dyes are examples. Suitable fluorophores are fluorine Fluorescein isothiocyanate (FITC), fluorescein Inthiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, C) y5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alex a555, Alexa 594, Alexa 647), near infrared (NIR) (700~900nm) fluorescent dyes, as well as carbocyanin and aminostyryl dyes. .

[0200] CARs, polypeptides, or proteins (functional portions and their functional components) as described herein A nucleic acid comprising a nucleotide sequence encoding any of the variants is described in this disclosure. It is provided as such.

[0201] A portion of the CAR containing an antibody or an antibody fragment may exist in various forms, in which case The antigen-binding domain is, for example, a single-domain antibody fragment (sdAb), scFv, and human It is expressed as part of a continuous polypeptide chain such as a chimeric antibody or a humanized antibody (Ha rlow et al., 1999, In: Using Antibodies:AL aboratory Manual,Cold Spring Harbor Labo ratory Press, NY; Harlow et al., 1989, In: Antibodies:A Laboratory Manual,Cold Spri ng Harbor, NY, Houston et al., 1988, Proc. Natl.Acad.Sci.USA 85:5879-5883;Bird et a l., 1988, Science 242:423426). In one embodiment, the CA of the present invention The antigen-binding domain of composition R contains an antibody fragment. In one embodiment, CAR contains scFv Contains antibody fragments.

[0202] The term "recombinant antibody" refers to, for example, antibodies expressed by bacteriophages or yeast expression systems. This term refers to antibodies produced using recombinant DNA technology, such as those expressed as antibodies. This DNA molecule, which is produced by the synthesis of the encoding DNA molecule, is an antibody protein. It should be interpreted as meaning an antibody that expresses an amino acid sequence that identifies the antibody, In that case, the DNA or amino acid sequence is available and known in the art. It is obtained using recombinant DNA or amino acid sequencing technology.

[0203] The term "antigen" refers to a molecule that triggers an immune response. This immune response involves antibody production. This may involve the activation of specific immunocompetent cells, or both. This means that virtually any macromolecule, including all proteins or peptides, can function as an antigen. They will understand that. Furthermore, antigens may be derived from recombinant or genomic DNA. If a person is a nucleotide, any DNA can encode a protein that triggers an immune response. The term is used herein by including a sequence or partial nucleotide sequence. It will be understood that the "antigen" is coded. Furthermore, a person skilled in the art will know that the antigen is, Understand that genes do not need to be encoded solely by their full-length nucleotide sequence. This disclosure involves the use of partial nucleotide sequences of two or more genes, but this does not include It is not limited to, and these nucleotide sequences can be arranged in various combinations. It is clear that this encodes polypeptides that induce a desired immune response.

[0204] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a signaling domain, and the extracellular antigen-binding domain is an anti-CD79b agent. Combine with the original.

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

[0206] In one embodiment, the extracellular antigen-binding domain is Heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of sequence number 14, and This includes the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) of sequence number 32. .

[0207] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence numbers 208, 216, 222, 228, 232, 238, 242, 248, 253 Select from the group consisting of , 257, 263, 268, and 274, and their conservative modifications. It contains a heavy chain CDR1 having the amino acid sequence, and the extracellular antigen-binding domain is CD79 Binds to the b antigen.

[0208] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence IDs 209, 217, 223, 233, 239, 243, 249, 254, 258 , 269, and 275, and amino acid compounds selected from the group consisting of these conservative modifications. It contains a heavy chain CDR2 with columns, and the extracellular antigen-binding domain binds to the CD79b antigen. .

[0209] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence IDs 210, 218, 224, 229, 234, 240, 244, 250, 255 Select from the group consisting of , 259, 264, 270, and 276, and their conservative modifications. It contains a heavy chain CDR3 having the amino acid sequence, and the extracellular antigen-binding domain is CD79 Binds to the b antigen.

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

[0212] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence numbers 211, 214, 215, 219, 225, 230, 235, 241, 245 , 251, 260, 265, 271, and 277, and the group consisting of their conservative modifications. It comprises a light chain CDR1 having an amino acid sequence selected from, and the extracellular antigen-binding domain is, Binds to the CD79b antigen.

[0213] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence IDs 212, 220, 226, 231, 236, 246, 261, 266, and 2 72, and a light chain C having an amino acid sequence selected from the group consisting of these conserved modifications. It contains DR2, and its extracellular antigen-binding domain binds to the CD79b antigen.

[0214] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Sequence IDs 213, 221, 227, 237, 247, 252, 256, 262, 267 , 273, or 278, and amino acid combinations selected from the group consisting of these conservative modifications. It contains a light chain CDR3 with columns, and the extracellular antigen-binding domain binds to the CD79b antigen. .

[0215] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR provides a transmission domain, and this extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, amino acid sequence of SEQ ID NO: 212 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, amino acid sequence of SEQ ID NO: 220 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 231 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, amino acid sequence of SEQ ID NO: 226 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, and a 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, amino acid sequence of SEQ ID NO: 246 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 252, and a 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, amino acid sequence of SEQ ID NO: 236 A light chain CDR2 having the amino acid sequence of SEQ ID NO. 256, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 262, and a 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, amino acid sequence of SEQ ID NO: 266 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, amino acid sequence of SEQ ID NO: 272 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 273, or Light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, amino acid sequence of SEQ ID NO: 266 It includes a light chain CDR2 having the amino acid sequence of SEQ ID NO: 278 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 this disclosure comprises an extracellular antigen-binding domain, a transmembrane domain, and The CAR provides an intracellular signaling domain, and this extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 Includes light chain CDR2 having the amino acid sequence of SEQ ID NO: 262 and 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) Amino acids of sequence numbers 274, 275, 276, 277, 266, and 278, respectively. Acid sequence, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CD Includes R2 and light chain CDR3.

[0218] In one embodiment, the extracellular antigen-binding domains are sequence numbers 257, 258, and 25, respectively. Heavy chain CDR1, heavy chain CDR2, heavy chain CDR1, heavy chain CDR2, heavy chain CDR1, have amino acid sequences 9, 260, 261, 262. Includes chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.

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

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

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

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

[0223] In one embodiment, the extracellular antigen-binding domain is Sequence ID 14 and 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 9 A heavy chain variable region containing an amino acid sequence with 9% sequence identity, and less than SEQ ID NO: 32. 50, at least 55, at least 60, at least 65, at least 70, less 75, at least 80, at least 85, at least 90, at least 95, less Both have 96%, at least 97%, at least 98%, or at least 99% sequence identity. It contains a light chain variable region that includes an amino acid sequence.

[0224] In one embodiment, the extracellular antigen-binding domain includes scFv. In several embodiments, scFv contains a linker polypeptide between the light chain variable region and the heavy chain variable region. In a specific embodiment, the extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 75-118. A selected amino acid sequence is included in the CD79b polypeptide (for example, the amino acids described herein). s cFv. In some embodiments, the linker polypeptide is sequence numbers 42-74. It contains an amino acid sequence selected from the group consisting of the following.

[0225] In one embodiment, the linker polypeptide contains the amino acid sequence of SEQ ID NO: 42. In the application form, the linker polypeptide is at least SEQ ID NOs. 42 and 50, and at least SEQ ID NOs. 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 , comprising an amino acid sequence having at least 98, or at least 99%, sequence identity.

[0226] In one embodiment, scFv is selected from the group consisting of sequence numbers 75 to 118. It contains an amino acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO 75. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 76. Fv contains the amino acid sequence of SEQ ID NO: 77. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 77 It contains an 8-amino acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 79 Includes. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 80. scFv contains the amino acid sequence of SEQ ID NO: 81. In one embodiment, scFv is distributed It contains the amino acid sequence of sequence number 82. In one embodiment, scFv is the amino acid sequence of sequence number 83. It contains an acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 84. In the application form, scFv contains the amino acid sequence of SEQ ID NO: 85. In one embodiment, scF v contains the amino acid sequence of SEQ ID NO: 86. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 87 It includes the amino acid sequence of SEQ ID NO: 88. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 88. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 89. scFv contains the amino acid sequence of SEQ ID NO: 90. In one embodiment, scFv is the sequence It contains the amino acid sequence number 91. In one embodiment, scFv is the amino acid sequence of sequence number 92. Includes a sequence. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 93. Morphologically, scFv contains the amino acid sequence of SEQ ID NO: 94. In one embodiment, scFv This includes the amino acid sequence of SEQ ID NO: 95. In one embodiment, scFv is the amino acid sequence of SEQ ID NO: 96 It includes an amino acid sequence. In one embodiment, scFv includes the amino acid sequence of SEQ ID NO: 97. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 98. In one embodiment, scFv contains the amino acid sequence of sequence number 99. In one embodiment, scFv is sequence number It contains the amino acid sequence of sequence number 100. In one embodiment, scFv is the amino acid sequence of sequence number 101. It contains an acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 102. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 103. cFv contains the amino acid sequence of sequence number 104. In one embodiment, scFv is sequence number It contains the amino acid sequence of number 105. In one embodiment, scFv is the amino acid sequence of number 106. It contains an acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 107. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 108. cFv contains the amino acid sequence of sequence number 109. In one embodiment, scFv is sequence number It contains the amino acid sequence of number 110. In one embodiment, scFv is the amino acid sequence of sequence number 111. It contains an acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 112. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 113. cFv contains the amino acid sequence of sequence number 114. In one embodiment, scFv is sequence number It contains the amino acid sequence of sequence number 115. In one embodiment, scFv is the amino acid sequence of sequence number 116. It contains an acid sequence. In one embodiment, scFv contains the amino acid sequence of SEQ ID NO: 117. In this embodiment, scFv contains the amino acid sequence of SEQ ID NO: 118.

[0227] In one embodiment, scFv is an array selected from the group consisting of sequence numbers 75 to 118. 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 It contains an amino acid sequence having the following characteristics. In one embodiment, scFv is at least the same as SEQ ID NO: 75 Also 50, at least 55, at least 60, at least 65, at least 70, and 75, at least 80, at least 85, at least 90, at least 95, and at least It also has 96%, at least 97%, at least 98%, or at least 99% sequence identity. , containing an amino acid sequence. In one embodiment, scFv is at least 50 of sequence number 76, 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, amino Contains an acid sequence. In one embodiment, scFv is at least sequence number 77 and 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, scFv includes at least sequence number 78 and 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In this embodiment, scFv is at least sequence number 79 and at least 50, at least 55, and Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, scFv is sequence number 80 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, s cFv is sequence number 81 and at least 50, at least 55, at least 60, and 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, scFv is , Sequence ID 82 and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, scFv is the sequence number No. 83 and at least 50, at least 55, at least 60, at least 65, and at least 70, at least 75, at least 80, at least 85, at least 90, and at least 95, at least 96, at least 97, at least 98, or at least 99% It contains an amino acid sequence having sequence identity. In one embodiment, scFv is sequence number 84 and 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 It contains an amino acid sequence having the following characteristics. In one embodiment, scFv is at least the same as sequence number 85. Also 50, at least 55, at least 60, at least 65, at least 70, and 75, at least 80, at least 85, at least 90, at least 95, and at least It also has 96%, at least 97%, at least 98%, or at least 99% sequence identity. , containing an amino acid sequence. In one embodiment, scFv is at least 50 of sequence number 86, 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, amino Contains an acid sequence. In one embodiment, scFv is at least sequence number 87 and 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, scFv includes sequence number 88 and 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In this embodiment, scFv is sequence number 89 and at least 50, at least 55, and Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, scFv is sequence number 90 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, s cFv is at least 50, at least 55, at least 60, and at least 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, scFv is , 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, scFv is the sequence number No. 93 and at least 50, at least 55, at least 60, at least 65, and at least 70, at least 75, at least 80, at least 85, at least 90, and at least 95, at least 96, at least 97, at least 98, or at least 99% It contains an amino acid sequence having column identity. In one embodiment, scFv is sequence number 94 and 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 It contains an amino acid sequence having the following characteristics. In one embodiment, scFv is at least the same as sequence number 95. Also 50, at least 55, at least 60, at least 65, at least 70, and 75, at least 80, at least 85, at least 90, at least 95, and at least It also has 96%, at least 97%, at least 98%, or at least 99% sequence identity. , including an amino acid sequence. In one embodiment, scFv is at least 50 of sequence number 96, 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, amino Contains an acid sequence. In one embodiment, scFv is at least sequence number 97 and 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, scFv includes at least sequence number 98 and 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In this embodiment, scFv is at least sequence number 99 and at least 50, at least 55, and Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, scFv is sequence number 100 and 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 comprising an amino acid sequence having at least 99% sequence identity. In one embodiment, scFv is sequence number 101 and at least 50, at least 55, at least 60, less At least 65, at least 70, at least 75, at least 80, at least 85, less at least 90, at least 95, at least 96, at least 97, at least 98, or It contains an amino acid sequence having at least 99% sequence identity. In one embodiment, scF v is at least 50, at least 55, at least 60, and 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 less Both contain amino acid sequences with 99% sequence identity. In one embodiment, scFv is Sequence ID 103 and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, scFv is the sequence number No. 104 and at least 50, at least 55, at least 60, at least 65, less 70, at least 75, at least 80, at least 85, at least 90, less 95%, at least 96%, at least 97%, at least 98%, or at least 99% It contains an amino acid sequence that has sequence identity. In one embodiment, scFv is sequence number 10 5 and at least 50, at least 55, at least 60, at least 65, at least 7 0, at least 75, at least 80, at least 85, at least 90, at least 9 5. At least 96, at least 97, at least 98, or at least 99% of the sequence It contains an amino acid sequence that is monomorphic. In one embodiment, scFv is less than SEQ ID NO: 106 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 It contains an amino acid sequence having the following characteristics. In one embodiment, scFv is less than SEQ ID NO: 107 50, at least 55, at least 60, at least 65, at least 70, less 75, at least 80, at least 85, at least 90, at least 95, less Both have 96%, at least 97%, at least 98%, or at least 99% sequence identity. It includes an amino acid sequence. In one embodiment, scFv is at least 5 of sequence number 108. 0, at least 55, at least 60, at least 65, at least 70, at least 7 5, at least 80, at least 85, at least 90, at least 95, at least 9 6. Having at least 97%, at least 98%, or at least 99% sequence identity, Contains a mino acid sequence. In one embodiment, scFv is at least 50 of sequence number 109, 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, amino acids having at least 97, at least 98, or at least 99% sequence identity. Includes an array. In one embodiment, scFv includes array numbers 110 and at least 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, scFv includes at least 50 and at least 55 of sequence number 111. , 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 , comprising an amino acid sequence having at least 98, or at least 99%, sequence identity. In one embodiment, scFv is sequence number 112 and at least 50, at least 55, and at least At least 60, at least 65, at least 70, at least 75, at least 80, less At least 85, at least 90, at least 95, at least 96, at least 97, at least It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In form, scFv is sequence number 113 and 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 It comprises an amino acid sequence having 98, or at least 99%, sequence identity. In one embodiment, scFv is sequence number 114 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, s cFv is at least 50, at least 55, at least 60, with sequence number 115. 65, at least 70, at least 75, at least 80, at least 85, less 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, scFv This is sequence number 116 and at least 50, at least 55, at least 60, at least 6 5, at least 70, at least 75, at least 80, at least 85, at least 9 0, at least 95, at least 96, at least 97, at least 98, or at least It also contains an amino acid sequence that has 99% sequence identity. In one embodiment, scFv is distributed Column number 117 and at least 50, at least 55, at least 60, at least 65, few 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 It contains an amino acid sequence having % sequence identity. In one embodiment, scFv is the sequence number 118 and at least 50, at least 55, at least 60, at least 65, and at least 70, at least 75, at least 80, at least 85, at least 90, and at least 95, at least 96, at least 97, at least 98, or at least 99% It contains amino acid sequences that have sequence identity.

[0228] In one embodiment, the extracellular antigen-binding domain is the same as that of SEQ ID NO: 113, or SEQ ID NO: 113 and 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 , an array of at least 96, at least 97, at least 98, or at least 99% It contains an amino acid sequence that is identical.

[0229] In one embodiment, the extracellular antigen-binding domain includes a signal polypeptide. In this embodiment, the signal polypeptide contains the amino acid sequence of SEQ ID NO: 37. In terms of morphology, the signal polypeptide is at least SEQ ID NOs. 37 and 50, at least SEQ ID NOs. 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity.

[0230] In one embodiment, the disclosure relates to an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. It provides a CAR containing a transmission domain, and the extracellular antigen-binding domain is SEQ ID NO: 119~ Contains an amino acid sequence selected from a group of 162. (Sequence codes 119-162) Another CAR having an extracellular antigen-binding domain, containing an amino acid sequence selected from the group. A key feature is that its extracellular antigen-binding domain binds to the CD79b antigen.

[0231] In one embodiment, the intracellular signaling domain is a TNF receptor superfamily The components include: chromium 9 (CD137), T cell surface glycoprotein CD3ζ chain (CD3z), and Differentiation cluster (CD27) component, differentiation cluster superfamily member (e.g., CD 28 or inducible T-cell co-stimulator (ICOS), etc.) It contains polypeptide components selected from the group consisting of components and combinations thereof.

[0232] In one embodiment, component CD137 includes the amino acid sequence of SEQ ID NO: 40. So, the CD137 component is at least SEQ ID NOs. 40 and 50, at least 55, at 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 It contains an amino acid sequence having 98, or at least 99%, sequence identity.

[0233] In one embodiment, the CD3z component includes the amino acid sequence of SEQ ID NO: 41. The CD3z component is at least 41, 50, 55, and 60 of sequence numbers. , 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 containing an amino acid sequence having at least 99% sequence identity.

[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 is sequence number 163 and 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 , having at least 97, at least 98, or at least 99% sequence identity, Contains an acid sequence.

[0235] In one embodiment, the transmembrane domain is a CD8a transmembrane region (CD8A-TM) polypeptide Contains cydore. In some embodiments, the CD8a-TM polypeptide is the same as in SEQ ID NO: 39 It contains an amino acid sequence. In one embodiment, the CD8a-TM polypeptide is sequence number 39 and 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 It contains an amino acid sequence having the following characteristics.

[0236] In one embodiment, the transmembrane domain is the α, β, or ζ chain of the T cell receptor, CD28, C D3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD13 7, or comprising at least (multiple) transmembrane regions of CD154. In another embodiment, The transmembrane domain consists of at least ζ, η, or FcεR1γ and β, MB1(Igα), B 29, or containing the transmembrane domain of CD3-γ, ζ, or η. In another embodiment, The transmembrane domain is synthetic, for example, leucine and valine, and phenylalanine. It contains mainly hydrophobic residues such as pretzels or tryptophan.

[0237] In one embodiment, the CAR has a transmembrane domain linked to an extracellular antigen-binding domain. It further includes a hinge region. In some embodiments, the hinge region is the 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 is at least 50 of sequence number 38, 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, amino acids having at least 97, at least 98, or at least 99% sequence identity. Includes an array. In some embodiments, the hinge region is the array EPKSCDKTHTCPP Contains CP (sequence number 285), or EPKSCDKTHTCPPCP (sequence number 285) 5) and 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% It contains amino acid sequences having sequence identity. In some embodiments, the hinge region is part of the sequence Contains ERKCCVECPPCP (sequence number 286) or ERKCCVECPPCP (Sequence number 286) and at least 50, at least 55, at least 60, at least 6 5, at least 70, at least 75, at least 80, at least 85, at least 9 0, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence with at least 99% sequence identity. In some embodiments, The region is the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP )3 (Sequence ID 287) is included, or ELKTPLGDTTHTCPRCP(EPKS CDTPPPCPRCP)3 (Sequence ID 287) and at least 50, at least 55, less 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, It contains an amino acid sequence with at least 98, or at least 99%, sequence identity. In some embodiments, the hinge region is array ESKYGPPCPSCP (sequence number 288) ) including or ESKYGPPCPSCP (SEQ ID NO: 288) and at least 50, less At least 55, at least 60, at least 65, at least 70, at least 75, less At least 80, at least 85, at least 90, at least 95, at least 96, less amino acids having at least 97, at least 98, or at least 99% sequence identity Includes arrays.

[0238] In one embodiment, the extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 119-162. The selected amino acid sequence is included. In one embodiment, the extracellular antigen-binding domain is sequence number It contains the amino acid sequence of number 119. In one embodiment, the extracellular antigen-binding domain is sequence number 119. It contains a sequence of 120 amino acids. In one embodiment, the extracellular antigen-binding domain is SEQ ID NO: 1 It contains a sequence of 21 amino acids. In one embodiment, the extracellular antigen-binding domain is sequence number 12. It contains the amino acid sequence 2. In one embodiment, the extracellular antigen-binding domain is sequence number 123. It contains the amino acid sequence of SEQ ID NO: 124. In one embodiment, the extracellular antigen-binding domain is SEQ ID NO: 124 It contains an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is the amino acid sequence of SEQ ID NO: 125. It contains a mino acid sequence. In one embodiment, the extracellular antigen-binding domain is the amine sequence of SEQ ID NO: 126. It contains an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is the amino acid sequence of SEQ ID NO 127. It contains an acid sequence. In one embodiment, the extracellular antigen-binding domain is the amino acid sequence of SEQ ID NO: 128. Includes sequence. In one embodiment, the extracellular antigen-binding domain is the amino acid sequence of SEQ ID NO: 129. Includes a column. In one embodiment, the extracellular antigen-binding domain is the amino acid sequence of SEQ ID NO: 130. It includes. In one embodiment, the extracellular antigen-binding domain has the amino acid sequence of SEQ ID NO: 131 Includes. 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 this embodiment, the extracellular antigen-binding domain includes the amino acid sequence of SEQ ID NO: 136. In the application, the extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 137. Morphologically, the extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 138. In this embodiment, the extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 139. In one embodiment, the extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 140. In one embodiment, the extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 141. The extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 142. In one embodiment, The extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 143. In one embodiment, The extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 144. In one embodiment, cells The extracellular antigen-binding domain contains the amino acid sequence of SEQ ID NO: 145. In one embodiment, extracellular The antigen-binding domain contains the amino acid sequence of SEQ ID NO: 146. In one embodiment, extracellular anti The primordial binding domain contains the amino acid sequence of SEQ ID NO: 147. In one embodiment, an extracellular antigen The binding domain contains the amino acid sequence of SEQ ID NO: 148. In one embodiment, extracellular antigen binding The combined domain contains the amino acid sequence of SEQ ID NO: 149. In one embodiment, it binds to extracellular antigens. The domain contains the amino acid sequence of SEQ ID NO: 150. In one embodiment, the extracellular antigen-binding domain The main component contains the amino acid sequence of SEQ ID NO: 151. In one embodiment, extracellular antigen-binding dormant The molecule contains the amino acid sequence of SEQ ID NO: 152. In one embodiment, extracellular antigen-binding domain The n contains the amino acid sequence of SEQ ID NO: 153. In one embodiment, the extracellular antigen-binding domain This includes the amino acid sequence of SEQ ID NO: 154. In one embodiment, the extracellular antigen-binding domain is , containing the amino acid sequence of SEQ ID NO: 155. In one embodiment, the extracellular antigen-binding domain is It contains the amino acid sequence of SEQ ID NO: 156. In one embodiment, the extracellular antigen-binding domain is distributed It contains the amino acid sequence of column number 157. In one embodiment, the extracellular antigen-binding domain is the sequence It contains amino acid sequence number 158. In one embodiment, the extracellular antigen-binding domain is sequence number 158. It contains the amino acid sequence of number 159. In one embodiment, the extracellular antigen-binding domain is sequence number 159. It contains a sequence of 160 amino acids. In one embodiment, the extracellular antigen-binding domain is SEQ ID NO: 1 It contains a sequence of 61 amino acids. In one embodiment, the extracellular antigen-binding domain is SEQ ID NO: 16 It contains the amino acid sequence 2.

[0239] In one embodiment, the extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 119-162. The selected array and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, extracellular antigen-binding dormancy The input is sequence number 119 and at least 50, at least 55, at least 60, and 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, it is an extracellular antigen The binding domain is sequence number 120 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, The extracellular antigen-binding domain is at least 50, at least 55, and is at least Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, the extracellular antigen-binding domain is sequence number 122 and 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In the embodiment, the extracellular antigen-binding domain is at least SEQ ID NO: 123 and 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, the extracellular antigen-binding domain is sequence number 124 and 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, amino It contains an acid sequence. In one embodiment, the extracellular antigen-binding domain is at least SEQ ID NO: 125 Also 50, at least 55, at least 60, at least 65, at least 70, and 75, at least 80, at least 85, at least 90, at least 95, and at least It also has 96%, at least 97%, at least 98%, or at least 99% sequence identity. , containing an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is sequence number 126 and 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 It comprises an amino acid sequence having the following characteristics: In one embodiment, the extracellular antigen-binding domain is the sequence number 127 and at least 50, at least 55, at least 60, at least 65, and at least 70, at least 75, at least 80, at least 85, at least 90, and at least 95, at least 96, at least 97, at least 98, or at least 99% It contains an amino acid sequence having serial identity. In one embodiment, the extracellular antigen-binding domain is Sequence ID 128 and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, extracellular antigen-binding dormancy The input is sequence number 129 and at least 50, at least 55, at least 60, and at least 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, it is an extracellular antigen The binding domain is sequence number 130 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, The extracellular antigen-binding domain is at least 50, at least 55, and is at least 131. Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, the extracellular antigen-binding domain is sequence number 132 and 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, It contains an amino acid sequence having at least 98, or at least 99%, sequence identity. In the embodiment, the extracellular antigen-binding domain is at least SEQ ID NO: 133 and 50, and at least 55, at least 60, at least 65, at least 70, at least 75, and at least 80, at least 85, at least 90, at least 95, at least 96, and at least The amino acid sequence has 97, at least 98, or at least 99% sequence identity. Includes. In one embodiment, the extracellular antigen-binding domain is sequence number 134 and 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, amino It contains an acid sequence. In one embodiment, the extracellular antigen-binding domain is at least SEQ ID NO: 135 Also 50, at least 55, at least 60, at least 65, at least 70, and 75, at least 80, at least 85, at least 90, at least 95, and at least It also has 96%, at least 97%, at least 98%, or at least 99% sequence identity. , containing an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is sequence number 136 and 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 It comprises an amino acid sequence having the following characteristics: In one embodiment, the extracellular antigen-binding domain is the sequence number 137 and at least 50, at least 55, at least 60, at least 65, and at least 70, at least 75, at least 80, at least 85, at least 90, and at least 95, at least 96, at least 97, at least 98, or at least 99% It contains an amino acid sequence having serial identity. In one embodiment, the extracellular antigen-binding domain is Sequence ID 138 and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, extracellular antigen-binding dormancy The input is sequence number 139 and at least 50, at least 55, at least 60, and 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, it is an extracellular antigen The binding domain is sequence number 140 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, The extracellular antigen-binding domain is at least SEQ ID NO: 141 and 50, at least 55, and Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at least 95, at least 96, at least 97, and at least It also includes an amino acid sequence having 98, or at least 99%, sequence identity. One embodiment So, the extracellular antigen-binding domain is sequence number 142 and 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, It contains amino acid sequences that have at least 98, or at least 99%, identical sequences. In the application form, the extracellular antigen-binding domain is at least SEQ ID NO: 143 and 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 It contains an amino acid sequence having 97, at least 98, or at least 99% sequence identity. In one embodiment, the extracellular antigen-binding domain is at least SEQ ID NO: 144 and 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, amino acids having at least 97, at least 98, or at least 99% sequence identity. Includes a sequence. In one embodiment, the extracellular antigen-binding domain is sequence number 145 and 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 Having 96, at least 97, at least 98, or at least 99% sequence identity, It contains an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is less than SEQ ID NO: 146. 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 It contains an amino acid sequence. In one embodiment, the extracellular antigen-binding domain is SEQ ID NO: 1 47 and 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% of the array It contains an amino acid sequence that is identical. In one embodiment, the extracellular antigen-binding domain is distributed Column numbers 148 and 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 9 It contains an amino acid sequence having 9% sequence identity. In one embodiment, extracellular antigen-binding dormancy The input is sequence number 149 and at least 50, at least 55, at least 60, and 65, at least 70, at least 75, at least 80, at least 85, and at least 90, at least 95, at least 96, at least 97, at least 98, or less It contains an amino acid sequence that has at least 99% sequence identity. In one embodiment, it is an extracellular antigen The binding domain is sequence number 150 and 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 it includes an amino acid sequence having at least 99% sequence identity. In one embodiment, The extracellular antigen-binding domain is at least 50, at least 55, and is at least Also 60, at least 65, at least 70, at least 75, at least 80, and at least 85, at least 90, at...

Claims

1. It is a chimeric antigen receptor (CAR), (a) an extracellular domain that specifically binds to the CD79b antigen, (b) Transmembrane domain and (c) Intracellular signaling domains optionally containing at least one co-stimulatory domain The CAR includes the above.

2. (d) CD8a - further including hinge region, The aforementioned transmembrane domain includes a CD8a transmembrane region (CD8A-TM) polypeptide. The intracellular signaling domain is TNF receptor superfamily member 9(C) D137) component-containing co-stimulatory domain and T cell surface glycoprotein CD3ζ chain (CD3z The CAR according to claim 1, comprising a primary signaling domain containing the component.

3. The CD8a hinge region has an amino acid sequence that is at least 90% identical to that of Sequence ID No.

38. Includes, The transmembrane domain contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

39. Mi, Kats / Or The intracellular signaling domain is at least 90% identical to that of Sequence ID No.

40. A co-stimulatory domain having an acid sequence and an amino acid sequence that is at least 90% identical to SEQ ID NO 41. The CAR according to claim 2, comprising a primary signaling domain having an acid sequence.

4. Including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain is Heavy chain complementarity determination region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 208, SEQ ID NO: A heavy chain CDR2 having a 209-amino acid sequence, and having the amino acid sequence of SEQ ID NO:

210. heavy chain CDR3, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 216, amino acid sequence of SEQ ID NO: 217 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 218, and a 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, amino acid sequence of SEQ ID NO: 223 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 224, and a 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, amino acid sequence of SEQ ID NO: 217 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 229, and a 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, amino acid sequence of SEQ ID NO: 233 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 234, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 238, amino acid sequence of SEQ ID NO: 239 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, and a 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, amino acid sequence of SEQ ID NO: 243 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 244, and a 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, amino acid sequence of SEQ ID NO: 249 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 250, and a 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, amino acid sequence of SEQ ID NO: 254 Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 255, Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 257, amino acid sequence of SEQ ID NO: 258 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 259, and a 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, amino acid sequence of SEQ ID NO: 243 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 264, and a 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, amino acid sequence of SEQ ID NO: 269 A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 270, or Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 274, amino acid sequence of SEQ ID NO: 275 It comprises a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 276 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

276. 、 The extracellular antigen-binding domain binds to the CD79b antigen, as described above.

5. The extracellular antigen-binding domain is a 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 amino acids of SEQ ID NO: 259 The CAR according to claim 4, comprising a heavy chain CDR3 having an array.

6. Including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, amino acid sequence of SEQ ID NO: 220 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, amino acid sequence of SEQ ID NO: 226 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 230, amino acid sequence of SEQ ID NO: 231 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 235, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, amino acid sequence of SEQ ID NO: 226 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 245, amino acid sequence of SEQ ID NO: 246 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 252, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 256, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 262, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 265, amino acid sequence of SEQ ID NO: 266 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, amino acid sequence of SEQ ID NO: 272 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 273, or Light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, amino acid sequence of SEQ ID NO: 266 It comprises a light chain CDR2 having the amino acid sequence of SEQ ID NO: 278 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, as described above.

7. The extracellular antigen-binding domain is a 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 amino acids of SEQ ID NO: 262 The CAR according to claim 6, comprising a light chain CDR3 having an arrangement.

8. The extracellular antigen-binding domain is Light chain CDR1 having the amino acid sequence of SEQ ID NO: 211, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 214, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 215, amino acid sequence of SEQ ID NO: 212 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 213, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 219, amino acid sequence of SEQ ID NO: 220 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, amino acid sequence of SEQ ID NO: 226 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 230, amino acid sequence of SEQ ID NO: 231 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 221, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 235, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 237, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 241, amino acid sequence of SEQ ID NO: 226 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 227, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 245, amino acid sequence of SEQ ID NO: 246 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 247, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 252, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 251, amino acid sequence of SEQ ID NO: 236 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 256, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 260, amino acid sequence of SEQ ID NO: 261 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 262, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 265, amino acid sequence of SEQ ID NO: 266 Light chain CDR2 having the amino acid sequence of SEQ ID NO: 267, Light chain CDR1 having the amino acid sequence of SEQ ID NO: 271, amino acid sequence of SEQ ID NO: 272 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 273, or Light chain CDR1 having the amino acid sequence of SEQ ID NO: 277, amino acid sequence of SEQ ID NO: 266 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 278, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 278, further A CAR as described in claim 4 or claim 5.

9. The extracellular antigen-binding domain is a 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 amino acids of SEQ ID NO: 262 The CAR according to claim 8, comprising a light chain CDR3 having an arrangement.

10. Including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain is sequence number 208. 、２１６、２２２、２２８、２３２、２３８、２４２、２４８、２５３、２５７、２６３ , heavy chain CDR1 having an amino acid sequence of 268 or 274, SEQ ID NOs: 209, 217, 223, 233, 239, 243, 249, 254, 258, 269, or 275 nets Heavy chain CDR2 with an acid sequence, SEQ ID NOs: 210, 218, 224, 229, 234, 2 amino acid sequences of 40, 244, 250, 255, 259, 264, 270, or 276 The heavy chain CDR3 contains sequence numbers 211, 214, 215, 219, 225, 230, and 23. It has an amino acid sequence of 5, 241, 245, 251, 260, 265, 271, or 277. Light chain CDR1, Sequence numbers 212, 220, 226, 231, 236, 246, 261 Light chain CDR2 having an amino acid sequence of 266 or 272, and SEQ ID NOs: 213, 22 1, 227, 237, 247, 252, 256, 262, 267, 273, or 278 The CAR comprises a light chain CDR3 having an amino acid sequence.

11. 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 IDs 268, 269, 270, 271, 272, and 273, respectively, or o) Amino acids of sequence numbers 274, 275, 276, 277, 266, and 278, respectively. Acid sequence, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CD A CAR according to any one of claims 1 to 10, comprising R2 and a light chain CDR3.

12. The extracellular antigen-binding domains are, respectively, sequence numbers 257, 258, 259, and 260. Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR have amino acid sequences 261 and 262.

3. Any one of claims 1 to 11, comprising light chain CDR1, light chain CDR2, and light chain CDR3. The CAR described in item 1.

13. Including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain is It contains an amino acid sequence that is at least 90% identical to a sequence selected from sequence numbers 1 to 18. heavy chain variable domain (VH), and / or An amino acid sequence that is at least 90% identical to a sequence selected from sequence numbers 19-36. It includes light chain variable domains (VLs), The extracellular antigen-binding domain binds to the CD79b antigen, as described above.

14. The extracellular antigen-binding domain is A heavy chain variable domain (VH) containing an amino acid sequence selected from SEQ ID NOs: 1 to 18, and / or Contains a light chain variable domain (VL) containing an amino acid sequence selected from SEQ ID NOs. 19-36. fruit, The extracellular antigen-binding domain binds to the CD79b antigen, as described in claim 13. R.

15. The extracellular antigen-binding domain is A heavy chain variable region containing an amino acid sequence that is at least 90% identical to Sequence ID No. 1, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to Sequence ID No. 1, and sequence Light chain variable region containing amino acid sequence number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to Sequence ID No. 2, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to Sequence ID No. 2, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to Sequence ID No. 3, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 20, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 4, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 19, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 5, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 22, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 5, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 23, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 6, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 24, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 7, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 26, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 8, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 25, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 9, and sequence A light chain variable region containing an amino acid sequence that is at least 90% identical to number 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 sequence number 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 sequence number 29, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 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 sequence number 31, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 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 sequence number 33, A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, and Light chain variable region containing an amino acid sequence that is at least 90% identical to sequence number 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 sequence number 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 sequence number 33, or A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, and A claim including a light chain variable region containing an amino acid sequence that is at least 90% identical to column number 36. The CAR described in item 13.

16. The extracellular antigen-binding domain is The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO:

19. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO:

20. Light chain variable region, A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain containing the amino acids of SEQ ID NO:

19. Chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO:

20. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO:

20. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO:

19. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO:

22. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO:

23. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO:

24. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO:

26. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 8, and the amino acid sequence of SEQ ID NO:

25. Light chain variable region, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO:

27. Light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 28 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of SEQ ID NO: 29 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12, and the amino acid sequence of SEQ ID NO: 30 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, and the amino acid sequence of SEQ ID NO: 31 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and the amino acid sequence of SEQ ID NO: 32 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15, and the amino acid sequence of SEQ ID NO: 33 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and the amino acid sequence of SEQ ID NO: 34 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 16, and the amino acid sequence of SEQ ID NO: 35 Including the light chain variable region, The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of SEQ ID NO: 33 Including light chain variable region, or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of SEQ ID NO: 36 A CAR according to any one of claims 13 to 15, comprising a light chain variable region.

17. 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: 14, and A claim including a light chain variable region containing an amino acid sequence that is at least 90% identical to column number 32. The CAR as described in item 13 or 15.

18. The extracellular antigen-binding domain is The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14, and the amino acid sequence of SEQ ID NO: 32 A CAR according to any one of claims 13 to 17, comprising a light chain variable region.

19. The extracellular antigen-binding domain comprises a single-stranded variable fragment (scFv), and the scFv is , comprising a heavy chain variable region (VH) and a light chain variable region (VL), any one of claims 1 to 18 The CAR as described.

20. The scFv has phosphorus between the heavy chain variable region (VH) and the light chain variable region (VL). The CAR according to claim 19, comprising a car polypeptide.

21. The linker polypeptide has an amino acid composition that is at least 90% identical to that of SEQ ID NO:

42. The CAR according to claim 20, including a column.

22. The linker polypeptide comprises the amino acid sequence of SEQ ID NO: 42, claim 20 or 2 CAR as described in 1.

23. The aforementioned scFv is an array selected from the group consisting of sequence numbers 75 to 118 and at least 9 A CAR according to any one of claims 19 to 22, comprising an amino acid sequence that is 0% identical.

24. The aforementioned scFv includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 75 to 118. , the CAR according to any one of claims 19 to 23.

25. The aforementioned scFv includes an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

113. A CAR according to any one of claims 19 to 23.

26. The scFv comprises the amino acid sequence of SEQ ID NO: 113, any one of claims 19 to 25. The CAR listed.

27. The extracellular antigen-binding domain comprises a signal polypeptide, according to any of claims 1 to 26. The CAR listed on one of the cards.

28. The signal polypeptide is at least 90% identical to the sequence of SEQ ID NO:

37. The CAR according to claim 27, comprising a no-acid sequence.

29. The signal polypeptide comprises the amino acid sequence of SEQ ID NO: 37, claim 27 or 2 CAR as described in 8.

30. The intracellular signaling domain is TNF receptor superfamily member 9(C) D137) component, T cell surface glycoprotein CD3ζ chain (CD3z) component, differentiation cluster ( CD27) component, differentiated cluster superfamily member component, and combinations thereof The claim according to any one of claims 4 to 29, comprising a polypeptide component selected from the group consisting of se. The CAR.

31. The aforementioned CD137 component is an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

40. The CAR according to claim 30, including a column.

32. The CD137 component comprises the amino acid sequence of SEQ ID NO: 40, as described in claim 30 or 31. Carrying a car.

33. The CD3z component is an amino acid sequence that is at least 90% identical to the sequence of Sequence ID No.

41. A CAR according to any one of claims 30 to 32, including the CAR according to any one of claims 30 to 32.

34. The CD3z component comprises the amino acid sequence of SEQ ID NO: 41, as per any of claims 30 to 33. The CAR as described in item 1.

35. The intracellular signaling domain is at least 90% identical to the sequence of Sequence ID No.

163. A CAR according to any one of claims 30 to 34, comprising a certain amino acid sequence.

36. Claim 3, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

163. A CAR as described in any one of items 0 to 35.

37. The aforementioned transmembrane domain includes a CD8a transmembrane region (CD8a-TM) polypeptide. A CAR according to any one of claims 4 to 36.

38. The CD8a-TM polypeptide is at least 90% identical to the sequence of Sequence ID No.

39. The CAR according to claim 37, comprising the amino acid sequence.

39. Claim 37, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:

39. Or the CAR as described in 38.

40. The transmembrane domain further comprises a hinge region that connects the transmembrane domain to the extracellular antigen-binding domain, A CAR according to any one of claims 4 to 39.

41. The CAR according to claim 40, wherein the hinge region is the CD8a hinge region.

42. The CD8a hinge region has an amino acid sequence that is at least 90% identical to that of Sequence ID No.

38. The CAR according to claim 41, including the following:

43. The CD8a hinge region comprises the amino acid sequence of SEQ ID NO: 38, as in claim 41 or 42. The CAR listed.

44. The extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 119 to 162. A amino acid sequence, or the amino acid sequence selected from the group consisting of SEQ ID NOs: 119 to 162. A CAR according to any one of claims 1 to 43, comprising an array having 90% identity with the column.

45. The extracellular antigen-binding domain is at least 90% identical to that of SEQ ID NO:

157. A CAR according to any one of claims 1 to 44, comprising an acid sequence.

46. The extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 157, claims 1 to 4. CAR as described in any of item 5.

47. The CAR is an amino acid sequence selected from the group consisting of SEQ ID NOs: 164 to 207, or The amino acid sequence selected from the group consisting of Sequence IDs 164 to 207 is 90% identical. A CAR according to any one of claims 1 to 46, comprising an array having the following characteristics.

48. The CAR contains an amino acid sequence that is at least 90% identical to SEQ ID NO:

202. A CAR as described in any of the requests 1 to 47.

49. The CAR includes the amino acid sequence of SEQ ID NO: 202, as described in any one of claims 1 to 48. Carrying a car.

50. Isolated lymphocytes expressing CAR according to any one of claims 1 to 49.

51. The isolated lymphocytes according to claim 50, wherein the lymphocytes are T lymphocytes.

52. The lymphocytes are natural killer (NK) cells, as isolated according to claim 51. Lymphocytes.

53. An isolated nucleic acid molecule encoding a CAR according to any one of claims 1 to 49.

54. A vector comprising the nucleic acid molecule described in claim 53.

55. A cell expressing the nucleic acid molecule described in claim 53.

56. An effective amount of lymphocytes according to any one of claims 50 to 52, and a pharmaceutically acceptable excipient. A pharmaceutical composition containing the following.

57. A method for treating a subject having cancer, wherein the method is A therapeutically effective amount of lymphocytes according to any one of claims 50 to 52, or according to claim 56. The process includes administering the listed pharmaceutical composition to the subject, thereby causing the lymphocytes to reach the subject. The method for inducing the death of cancer cells in [a specific location].

58. The method according to claim 57, wherein the cancer is B-cell lymphoma.

59. The method according to claim 57 or claim 58, wherein the cancer is non-Hodgkin lymphoma.

60. The aforementioned cancers are diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma. (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia ( ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), mucosal-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or The method according to any one of claims 57 to 59, wherein the patient is a plasmacytoma.

61. A method for targeting and killing cancer cells, wherein the method is This includes bringing the cancer cells into contact with the lymphocytes described in any one of claims 50 to 52. The method wherein the lymphocytes thereby induce the death of the cancer cells.

62. The method according to claim 61, wherein the cancer cells are malignant B cells.

63. The cancer cells are non-Hodgkin lymphoma cells, as described in claim 61 or claim 62. The method.

64. The aforementioned cancer cells are diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma. Parkinson's disease (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZ), acute lymphoblastic leukemia Disease (ALL), Chronic Lymphocytic Leukemia (CLL), Multiple Myeloma (MM), Mucosal-Associated Lymph Tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, The method according to any one of claims 61 to 63, wherein the cells are plasmacytoma cells.

65. A method for detecting the presence of cancer in a subject, The cell sample obtained from the subject is brought into contact with the CAR according to any one of claims 1 to 49. By causing this, a CAR-cell complex is formed, To detect the complex (wherein the detection of the complex is the presence of cancer in the subject) The method includes (indicating presence).

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