Chimeric antigen receptors based on single-domain antibodies, and methods of use thereof

Abstract

To provide chimeric antigen receptors based on single-domain antibodies, and methods of use thereof.SOLUTION: The present application provides single-domain antibodies, and chimeric antigen receptors comprising one or more antigen binding domains each comprising a single-domain antibody. Further provided are engineered immune effector cells (such as T cells) comprising the chimeric antigen receptors. Pharmaceutical compositions, kits and methods of treating cancer are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a copy of Chinese Patent Application No. CN201510490 filed on August 11, 2015. 002.8, and Chinese Patent Application No. CN201510 filed on November 2, 2015 This application claims the benefit of U.S. Provisional Patent Application No. 733585.2, the contents of which are incorporated herein by reference. are incorporated herein in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety. Incorporated into: Sequence Listing in Computer Readable Format (CRF) (Filename: 761422 000340SEQLISTING.txt, Recording date: August 9, 2016, Size: 3 55KB).

[0003] Technical field of the invention The present invention relates to single domain antibodies, chimeric antigen receptors, engineered immune effector cells, The present invention further relates to chimeric antigen receptors, particularly chimeric antigen receptors, for therapeutic use. Cell activation and expansion for receptor-based T cell immunotherapy. [Background technology]

[0004] With the development of tumor immunotherapy and clinical techniques, chimeric antigen receptor T cell (CAR-T) immunotherapy Immunotherapy is currently one of the most promising tumor immunotherapy approaches. Carrier antigen receptors (CARs) consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a null transduction domain. The extracellular antigen-binding domain targets a specified tumor antigen. The CAR may comprise a single chain variable fragment (scFv) that binds to the CAR. Using immunoprecipitation techniques, these antibodies can be expressed on the surface of T cells. They bind to target tumor antigens. CARs then bind to available major histocompatibility complex (MHC) receptors specific for the target tumor antigen. It is thought that the antibody will initiate a specific anti-tumor response in an antigen-dependent manner, without gender restriction. In addition, it can activate T cells.

[0005] Single domain antibodies (sdAbs) are characterized by their ability to bind to a single monomeric antibody variable domain. , which differ from conventional four-chain antibodies. For example, camelids and sharks produce heavy chain-only antibodies (HcAbs). They produce a single domain antibody, named , which naturally lacks light chains. The antigen-binding fragment in each arm of a chain-only antibody consists of a single heavy chain variable domain (V H H), which can have high affinity for antigen without the assistance of light chains. Camelidae V H H is the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD. It is known as.

[0006] All publications, patents, patent applications, and published patent applications referenced herein , the disclosures of which are incorporated herein by reference in their entireties. Summary of the Invention

[0007] This application relates to single domain antibodies, single domain antibody-based chimeric antigen receptors (CARs), (V H H fragments, engineered immune effector cells, and cancer immunotherapy Its usage is provided.

[0008] An embodiment of the present application provides an anti-CD19 sdAb comprising the CDR regions of SEQ ID NO:76. In some embodiments, the anti-CD19 sdAb comprises the amino acid sequence of SEQ ID NO: 1. CDR1 comprising the amino acid sequence of SEQ ID NO: 2, and CDR2 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD19 sdAb comprises a CDR3 comprising the sequence of SEQ ID NO: 76 V containing the amino acid sequence H Contains the H domain.

[0009] In some embodiments, an anti-CD19 heavy chain only antibody (HcAb), or one of the antibodies described above, is used. Antigen binding proteins comprising any one of the anti-CD19 sdAbs are provided.

[0010] Aspects of the present application include: (a) an anti-CD19 sdAb (as described above) (b) an extracellular antigen-binding domain comprising a transmembrane domain; (c) an intracellular signaling domain. In some embodiments, the CAR is monospecific. R is monovalent. In some embodiments, the CAR is multivalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (e.g., bispecific).

[0011] An embodiment of the present application provides an anti-CD20 sdAb comprising the CDR regions of SEQ ID NO:77. In some embodiments, the anti-CD20 sdAb comprises the amino acid sequence of SEQ ID NO:4. CDR1 comprising the amino acid sequence of SEQ ID NO:5, and CDR2 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-CD20 sdAb comprises a CDR3 comprising the sequence of SEQ ID NO: 77. V containing the amino acid sequence H Contains the H domain.

[0012] In some embodiments, an anti-CD20 heavy chain only antibody (HCAB), or any of the antibodies described above, is used. Antigen binding proteins comprising any one of the anti-CD20 sdAbs are provided.

[0013] Aspects of the present application include: (a) an anti-CD20 sdAb (as described above) (b) an extracellular antigen-binding domain comprising a transmembrane domain; (c) an intracellular signaling domain. In some embodiments, the CAR is monospecific. R is monovalent. In some embodiments, the CAR is multivalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (e.g., bispecific).

[0014] An embodiment of the present application is an anti-BC antibody comprising any one of the CDR regions of SEQ ID NOs: 78 to 88. In some embodiments, the anti-BCMA sdAb is: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 18 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 29; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 19 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 20 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and CDR2 comprising the amino acid sequence of SEQ ID NO: 21 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 32; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 22 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 33; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 12 and the amino acid sequence of SEQ ID NO: 23 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 24 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 25 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 36; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 26 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 37; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, and CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38; or (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 17, and CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. include. In some embodiments, the anti-BCMA sdAb is selected from the group consisting of SEQ ID NOs: 78-88. V containing the selected amino acid sequence H Contains the H domain.

[0015] In some embodiments, an anti-BCMA heavy chain only antibody (HCAB), or any of the antibodies described above, is used. Antigen binding proteins comprising any one of the anti-BCMA sdAbs are provided.

[0016] Aspects of the present application include: (a) an anti-BCMA sdAb (as described above); (b) an extracellular antigen-binding domain comprising a transmembrane domain; (c) an intracellular signaling domain. In some embodiments, the CAR is monospecific. R is monovalent. In some embodiments, the CAR is multivalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (e.g., bispecific).

[0017] An embodiment of the present application is an anti-C antibody comprising any one of the CDR regions of SEQ ID NOs: 89 to 100. In some embodiments, the anti-CD38 sdAb is : (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, and CDR2 comprising the amino acid sequence of SEQ ID NO: 52 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 64; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 41, and CDR2 comprising the amino acid sequence of SEQ ID NO: 53 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 42, and CDR2 comprising the amino acid sequence of SEQ ID NO: 54 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO: 55 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 67; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, and CDR2 comprising the amino acid sequence of SEQ ID NO: 56 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 45 and the amino acid sequence of SEQ ID NO: 57 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 69; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 58 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 70; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 47, and CDR2 comprising the amino acid sequence of SEQ ID NO: 59 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 48, and CDR2 comprising the amino acid sequence of SEQ ID NO: 60 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 49, and CDR2 comprising the amino acid sequence of SEQ ID NO: 61 and CDR3 comprising the amino acid sequence of SEQ ID NO: 73; (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 50, and CDR2 comprising the amino acid sequence of SEQ ID NO: 62 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74; or (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 51, and CDR2 comprising the amino acid sequence of SEQ ID NO: 63 and CDR3 comprising the amino acid sequence of SEQ ID NO: 75. include. In some embodiments, the anti-CD38 sdAb is selected from the group consisting of SEQ ID NOs: 89-100. V comprising an amino acid sequence selected from H Contains the H domain.

[0018] In some embodiments, an anti-BCMA heavy chain only antibody (HCAB), or any of the antibodies described above, is used. Antigen binding proteins comprising any one of the anti-BCMA sdAbs are provided.

[0019] Aspects of the present application include: (a) an anti-CD38 sdAb (as described above); (b) an extracellular antigen-binding domain comprising a transmembrane domain; (c) an intracellular signaling domain. In some embodiments, the CAR is monospecific. R is monovalent. In some embodiments, the CAR is multivalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (e.g., bispecific).

[0020] An embodiment of the present application is a method for detecting an extracellular antigen-binding domain comprising: (a) an anti-CD22 sdAb; and (b) (c) a CD22 chimeric antibody comprising: In some embodiments, the CAR is monospecific. In some embodiments, the CAR is monovalent. In some embodiments, the CAR is multivalent (bivalent). In some embodiments, the CAR is multispecific (bispecific, trivalent, etc.). etc.).

[0021] An embodiment of the present application provides a method for the preparation of a first single domain antibody (sd) that specifically binds to a first antigen. and a second single domain antibody (sdAb) that specifically binds to a second antigen. (b) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. and a polypeptide comprising the chimeric antigen receptor (CAR). In some embodiments, the first sdAb is located N-terminal to the second sdAb. In this embodiment, the first sdAb is positioned C-terminal to the second sdAb.

[0022] In some embodiments according to any one of the CARs provided above, the first antibody The primary and secondary antigens are CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, E from GFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77 In some embodiments, the first sdAb is selected from the group consisting of B The antibody is an anti-BCMA sdAb, such as any one of the CMA sdAbs. In embodiments, the CAR comprises at least two copies (2, 3, or In some embodiments, the extracellular antigen-binding domain comprises one or more copies of the nucleotide sequence (e.g., nucleotide sequence 1, nucleotide sequence 2, nucleotide sequence 3, nucleotide sequence 4, nucleotide sequence 5, nucleotide sequence 6, nucleotide sequence 7, nucleotide sequence 8, nucleotide sequence 9, nucleotide sequence 10, nucleotide sequence 11, nucleotide sequence 12, nucleotide sequence 13, nucleotide sequence 14, nucleotide sequence 15, nucleotide sequence The first sdAb may be an anti-CD19 sdAb, such as any one of the anti-CD19 sdAbs described above. In some embodiments, the first sdAb is a CD19 sdAb. The anti-CD20 sdAb is any one of the anti-CD20 sdAbs listed in In some embodiments, the first sdAb is any of the anti-CD38 sdAbs described above. In some embodiments, the CAR is an anti-CD38 sdAb, such as any one of Contains at least two copies (e.g., 2, 3, or more copies) of the D38 sdAb In some embodiments, the first sdAb comprises an extracellular antigen-binding domain comprising an anti-CD 22 sdAb.

[0023] In some embodiments according to any one of the CARs provided above, the first antibody The antigen is different from the second antigen. In some embodiments, the CAR is multispecific, such as bispecific. In some embodiments, the first sdAb is an anti-BCMA sdAb. and the second sdAb is an anti-CD38 sdAb. In some embodiments, the first One sdAb is an anti-BCMA sdAb and the second sdAb is an anti-CD19 sdAb In some embodiments, the first sdAb is an anti-CD19 sdAb and the second In some embodiments, the first sdAb is an anti-CD20 sdAb. One sdAb is an anti-CD19 sdAb and the second sdAb is an anti-CD22 sdAb.

[0024] In some embodiments according to any one of the monospecific CARs provided above, In some embodiments, the first antigen is the same as the second antigen. In some embodiments, the CAR is bivalent or In some embodiments, the first and second sdAb are the same or trivalent. In some embodiments, the first sdAb specifically binds to an epitope of the second sdAb. In some embodiments, the first sdAb and the second sdAb are the same as They specifically bind to different epitopes.

[0025] In some embodiments according to any one of the CARs provided above, the first s The dAb and / or the second sdAb may be camelid, chimeric, human or humanized.

[0026] In some embodiments according to any one of the CARs provided above, the first s The dAb and the second sdAb are directly fused to each other via a peptide bond. In embodiments, the first and second sdAb are linked to each other via a peptide linker. In some embodiments, the peptide linker is about 50 or less (about any A single amino acid (e.g., 35, 25, 20, 15, 10, or 5 or less) in length. In some embodiments, the peptide linker is an amino acid selected from SEQ ID NOs: 144-151. Contains the acid sequence.

[0027] CARs provided above (CD19 CAR, CD20 CAR, BCMA CAR, C Several CARs according to one of the following: In embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80 , CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8 or CD28. In embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 132 or SEQ ID NO: 133. include.

[0028] CARs provided above (CD19 CAR, CD20 CAR, BCMA CAR, C Several CARs according to one of the following: In embodiments, the intracellular signaling domain is a member of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain The reach domain is derived from CD3ζ. In some embodiments, the reach domain is derived from CD3ζ. The domain comprises the amino acid sequence of SEQ ID NO:140 or SEQ ID NO:141.

[0029] CARs provided above (CD19 CAR, CD20 CAR, BCMA CAR, C Several CARs according to one of the following: In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of: The antibody contains the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137. In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 136 and / or SEQ ID NO: 137. Contains the amino acid sequence of sequence number 137.

[0030] CARs provided above (CD19 CAR, CD20 CAR, BCMA CAR, C Several CARs according to one of the following: In embodiments, the CAR comprises an extracellular antigen-binding domain C-terminal and a transmembrane domain N-terminal. In some embodiments, the hinge domain further comprises a hinge domain located between is derived from CD8α. In some embodiments, the hinge domain is SEQ ID NO: 130 It contains the amino acid sequence of

[0031] In some embodiments, the CAR comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide further comprises a signal peptide selected from the group consisting of CD8α, GM-CS, F receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. The null peptide comprises the amino acid sequence of SEQ ID NO:127.

[0032] Aspects of the present application provide any one of the chimeric antigen receptors listed in Tables 4, 5, and 6. In some embodiments, the CAR is selected from the group consisting of SEQ ID NOs: 152-174, 198-201, , 206-216, 248-249, 257-260, and 265-270 The amino acid sequence includes an amino acid sequence selected from the group consisting of:

[0033] The present application relates to SEQ ID NOs: 76-100, 152-174, 198-201, 206- 216, 248-249, 257-260, and 265-270 The present invention provides a polypeptide comprising an amino acid sequence of

[0034] Aspects of the present application include the CARs provided above (CD19 CAR, CD20 CAR, BC Any one of the following: MA CAR, CD38 CAR, and CD22 CAR In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding The amino acid sequences are SEQ ID NOs: 175-197, 202-205, 217-227, and 250-251. , 261-264, and 271-276. wherein the isolated nucleic acid further comprises a second nucleic acid sequence encoding a second CAR, The nucleic acid sequence encoding R may contain a self-cleaving peptide such as a T2A, P2A, or F2A peptide. The second nucleic acid sequence is operably linked to the second nucleic acid sequence via a third nucleic acid sequence encoding the In some embodiments, the third nucleic acid sequence is SEQ ID NO: 256. In some embodiments, the isolated nucleic acid is a DNA molecule. , an RNA molecule.

[0035] An embodiment of the present application is a vector comprising any one of the isolated nucleic acids described above. In some embodiments, the vector is an expression vector. In embodiments, the vector is a viral vector. is a lentiviral vector.

[0036] Aspects of the present application include the CARs provided above (CD19 CAR, CD20 CAR, BC Any one of the following: MA CAR, CD38 CAR, and CD22 CAR or any one of the isolated nucleic acids described above, or the vector described above. The present invention provides engineered immune effector cells comprising any one of the following: In some embodiments, the engineered immune effector cells comprise two or more Cs described above. AR(CD19 CAR, CD20 CAR, BCMA CAR, CD38 CAR, and and CD22 CARs), and the two or more CARs are directed to different antigens. In some embodiments, the immune effector cells are selected from the group consisting of T cells, NK cells, and IL-1 cells. cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells In some embodiments, the immune effector cells are T cells.

[0037] Aspects of the present application include any one of the engineered immune effector cells described above. and a pharmaceutically acceptable carrier. Further provided is a method of treating a subject, comprising administering to an individual an effective amount of any of the pharmaceutical compositions described above. In some embodiments, the method comprises administering one of the engineered immune effectors In some embodiments, the cells are autologous. In some embodiments, the cells are engineered immune effector cells. In some embodiments, the cancer is a liquid cancer. In some cases, the cancer is multiple myeloma, acute lymphocytic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is a solid cancer, such as glioblastoma.

[0038] An embodiment of the present application is directed to the use of the anti-CD19 sdAb, anti-CD20 sdAb, anti-C D38 sdAb, or anti-BCMA sdAb, and a pharmaceutically acceptable carrier. and a carrier adapted to administer the compound. In some embodiments, the compound is administered to an individual. Further provided is a method of treating a disease, such as cancer, comprising administering to an individual an effective amount of a pharmaceutical composition. This includes:

[0039] The single domain antibodies, CARs, engineered immune effector cells, isolated Also disclosed are methods of use, kits, and articles of manufacture comprising any one of the nucleic acids or vectors described above. Provided. [Brief explanation of the drawings]

[0040] [Figure 1A] The structure of a VHH-based CAR is compared with that of a conventional scFv-based CAR. The schematic structure on the left shows an exemplary monospecific monovalent CAR with an extracellular antigen-binding domain comprising a VHH domain. The schematic structure on the right shows an exemplary monospecific monovalent CAR with an extracellular antigen-binding domain comprising an scFv domain. [Figure 1B] The structure of a VHH-based CAR with two antigen binding sites is compared with the structure of a conventional scFv-based CAR with two antigen binding sites. The schematic structure on the left is an exemplary CAR with an extracellular antigen binding domain comprising two VHH domains. The two VHH domains can be the same or different. The schematic structure on the right shows an exemplary CAR with an extracellular antigen binding domain comprising two scFv domains. The two scFv domains can be the same or different. [Figure 1C]Schematic structures of exemplary bivalent and bispecific VHH-based CARs are shown. The schematic structure in the top left panel shows an exemplary monospecific, bivalent CAR with an extracellular antigen-binding domain comprising two identical VHH domains, each of which specifically binds epitope 1 of antigen A. The schematic structure in the top right panel shows an exemplary monospecific, bivalent CAR with an extracellular antigen-binding domain comprising a first VHH domain that specifically binds epitope 1 of antigen A and a second VHH domain that specifically binds epitope 2 of antigen A. Epitope 1 and epitope 2 of antigen A may differ in their structure and / or sequence. The schematic structure in the bottom left panel shows an exemplary bispecific CAR with an extracellular antigen-binding domain comprising a first VHH domain that specifically binds antigen A and a second VHH domain that specifically binds antigen B. Antigen A and antigen B are different antigens. [Figure 1D] 1 shows the schematic structure of an exemplary VHH-based CAR that has three or more VHH domains.CAR can have multiple VHH domains that are fused directly to each other or via a peptide linker.VHH domains can be the same or different.Different VHH domains can specifically bind to different epitopes on the same antigen or different antigens. [Figure 1E]

[0023] Figure 1 shows exemplary engineered immune effector cells co-expressing two different VHH-based CARs. The exemplary engineered immune effector cells in the left panel co-express two different monospecific, monovalent CARs. The exemplary engineered immune effector cells in the middle panel co-express a monospecific, monovalent CAR and a bispecific or bivalent CAR. The exemplary engineered immune effector cells in the right panel co-express two different bispecific or bivalent CARs. The CARs may recognize different antigens. [Figure 2A] 1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs comprising various anti-BCMA (i.e., anti-CD269) or anti-CD38 single domain antibodies against the multiple myeloma cell line RPMI8226.Luc. [Figure 2B]1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs comprising various anti-BCMA (i.e., anti-CD269) or anti-CD38 single domain antibodies against the glioblastoma cell line U87MG.Luc. [Figure 3A] 1 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bispecific CAR against the multiple myeloma cell line RPMI8226.Luc. [Figure 3B] 1 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bispecific CAR against the multiple myeloma cell line RPMI8226.Luc. [Figure 4] 1 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bispecific CAR against the multiple myeloma cell line RPMI8226.Luc. [Figure 5] 1 shows the construction of an exemplary bispecific CAR targeting CD19 and CD20, an exemplary monospecific CAR targeting CD19, and an exemplary monospecific CAR targeting CD20. [Figure 6] 1 shows the results of in vitro cytotoxicity assays of various T cells. The upper left panel shows the results of untransduced control T cells. The upper right panel shows the results of T cells expressing an exemplary CD19 CAR. The lower left panel shows the results of T cells expressing an exemplary CD20 CAR. The lower right panel shows the results of T cells expressing an exemplary bispecific CD19 x CD20 CAR. [Figure 7] 1 shows the results of an in vivo anti-tumor assay of T cells expressing an exemplary bispecific CAR targeting CD19 and CD20. [Figure 8A]

[0023] Figure 1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific, bivalent CARs against the multiple myeloma cell line RPMI8226.Luc. The CARs each contain an extracellular antigen-binding domain containing two different anti-BCMA (i.e., anti-CD269) sdAbs. [Figure 8B]

[0033] Figure 1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific, bivalent CARs against the glioblastoma cell line U87MG.Luc. The CARs each contain an extracellular antigen-binding domain containing two different anti-BCMA (i.e., anti-CD269) sdAbs. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present application provides a method for the production of single domain antibodies (sdAbs) containing extracellular antigen-binding domains. Monospecific, multispecific (e.g., bispecific), and multivalent (e.g., bivalent or trivalent) chimeric antigen reception Unlike antigen-binding fragments derived from conventional four-chain antibodies, sdAbs are V H sdAbs only contain a single variable domain such as H. Therefore, sdAbs are It is more efficient than antigen-binding fragments such as scFv, which are currently used as extracellular antigen-binding domains. Furthermore, the pairing of heavy and light chains is required during the folding of sdAb. Therefore, misfolding of the extracellular antigen-binding domain may be a contributing factor to the failure of sdAb-based CARs. This can be reduced in engineered immune cells that express a different epitope or antigen. Multiple copies of the target sdAb or an extracellular antigen-binding domain containing multiple sdAbs CARs having the above structure can be conveniently constructed and recombinantly produced, thereby enabling multivalent and multivalent CARs. Providing an efficient platform for the preparation and screening of bispecific CARs Furthermore, the small footprint of sdAbs allows for the identification of hidden antigen targets and antigens in tumor tissue. This may allow the CAR access to the pitope.

[0042] Multispecific and multivalent CARs are an improvement over monospecific monovalent CARs for cancer immunotherapy Cancer cells are generally unstable, which means that they may have increased efficacy in targeting the target antigen. escape from targeted therapy by mutating or losing the gene that encodes By targeting two or more different epitopes or antigens on cancer cells, Therefore, multivalent or multispecific CARs allow cancer cells to express engineered immune effectors that express the CAR. This may make it more difficult for the immune system to completely escape targeting by other immune cells (e.g., T cells). The small size of the extracellular antigen-binding domains allows for the multivalent or multispecific CARs of the present application to be easily identified. The tandem-fused single domain antibodies used as the main The functional integrity and binding affinity to the target antigen can be maintained, thereby enhancing the efficacy of CAR-mediated Multivalent or multispecific CARs allow for effective targeting of each epitope or antigen. express or co-express two or more chimeric antigen receptors that target different tumor antigens. The engineered immune effector cells exhibit abnormalities in protein-antigen processing and presentation. This may overcome the underlying tumor immune escape mechanisms.

[0043] Thus, aspects of the present application include: (a) a first single domain antibody that specifically binds to a first antigen; a single domain antibody (sdAb) and a second single domain antibody (sdAb) that specifically binds to a second antigen. (b) an extracellular antigen-binding domain comprising (b) a transmembrane domain; and (c) an intracellular signaling domain. a multispecific (e.g., bispecific) chimeric antigen comprising a polypeptide comprising a transduction domain; In some embodiments, the first antigen is different from the second antigen. In the example, the first antigen is BCMA and the second antigen is CD38. In some embodiments, the first antigen is CD19 and the second antigen is BCMA. In an embodiment, the first antigen is CD19 and the second antigen is CD20. In some embodiments, the first antigen is CD19 and the second antigen is CD22.

[0044] In another embodiment, (a) a plurality of single domain antibodies (sdAbs) that specifically bind to an antigen (b) an extracellular antigen-binding domain comprising a transmembrane domain; and (c) an intracellular signaling domain. and a multivalent chimeric antigen receptor (CAR) comprising a polypeptide comprising: .

[0045] In another embodiment, (a) a first single domain that specifically binds to a first epitope of an antigen. Cells comprising the antibody and a second single domain antibody that specifically binds to a second epitope of the antigen. (b) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. A multivalent chimeric antigen receptor (CAR) is provided, comprising a polypeptide comprising a first antigen receptor (AAR) and a second antigen receptor (TAR), The epitope is different from the second epitope.

[0046] Novel anti-CD19, anti-CD20, anti-BCMA, and anti-CD38 single domain antibodies Further provided is a chimeric antigen receptor comprising any one of said sdAbs.

[0047] Engineered immune effector cells (e.g., T cells) containing CARs, engineered immune effectors Pharmaceutical compositions, kits, products for treating cancer using tumor cells or single domain antibodies, and methods are also described herein.

[0048] I. Definition The practice of the present invention is within the skill of those in the art to develop and implement viral vectors, unless specifically indicated to the contrary. These are performed using conventional methods of biology, immunology, microbiology, molecular biology, and recombinant DNA technology. Many of these techniques are described below for illustrative purposes. Such techniques are well explained in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunolo gy, John Wiley & Sons, New York, NY(2009) ;Ausubel et al,Short Protocols in Molecule lar Biology,3 rd ed.,Wiley & Sons,1995;Sa mbrook and Russell,Molecular Cloning:AL aboratory Manual(3rd Edition,2001 );Mani atis et al.Molecular Cloning:A Laboratory y Manual(1982);DNA Cloning:A Practical A pproach,vol.I & II(D.Glover,ed.);Oligonu Cleotide Synthesis(N.Gait,ed.,1984);Nucl eic Acid Hybridization(B.Hames & S.Higgi ns, eds., 1985);Transcription and Translat ion(B.Hames & S.Higgins, eds.,1984);Anima l Cell Culture(R.Freshney,ed.,1986);Perb al,A Practical Guide to Molecular Clonin g (1984), and other similar references.

[0049] The term "antibody" refers to a monoclonal antibody (a full-length 4-antibody having an immunoglobulin Fc region). antibody compositions with polyepitopic specificity, polyclonal antibodies, and polyclonal antibodies. Multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as anti- Immunoglobulins include antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." Antibodies contemplated herein include single domain antibodies, such as heavy chain only antibodies.

[0050] The basic four-chain antibody unit consists of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are heterotetrameric glycoproteins composed of five basic heterotetrameric units. In addition to the 5-mer unit, it consists of an additional polypeptide called the J chain, which contains 10 antigen-binding domains. IgA antibodies contain 2 to 5 basic four-chain units. This unit is polymerized to form multivalent assemblies combined with J chains. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each light chain is linked to a heavy chain by one covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. The H chain has a variable domain (V H ) followed by 3 for each of the α and γ chains Two constant domains (C H ), and four C for μ and ε isotypes H Domain Each L chain has a variable domain (V L ), followed by the opposite end It has a constant domain in V. L is V H is consistent with C L is the first constant domain of the heavy chain In (C H 1) The specific amino acid residues in the light chain variable domain and the heavy chain variable domain are consistent with It is believed that an interface is formed between the V H and V L By pairing together The structures and properties of different classes of antibodies are described in detail in the For example, Basic and Clinical Immunology,8th Edi tion,Daniel P.Sties,Abba I.Terr and Tris tram G.Parsolw(eds),Appleton & Lange,Nor See Walk, Conn., 1994, p. 71 and chapter 6. The derived light chains are classified as kappa and lambda based on the amino acid sequence of their constant domains. These heavy chain constants can be assigned to one of two distinct classes called Domain (C H Depending on the amino acid sequence of the nucleotides, immunoglobulins are divided into different classes or Immunoglobulins can be assigned to different types: IgA, IgD, IgE, IgG, There are five classes of IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes have C H Based on relatively minor differences in sequence and function For example, humans have the following subclasses: IgG1, IgG2A , IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0051] The term "heavy chain only antibody" or "HCAb" refers to an antibody that contains a heavy chain but not the heavy chain typically found in four-chain antibodies. It refers to a functional antibody that lacks the light chains expressed by camelids (camels, llamas, or apes). Lupaka et al.) are known to produce HCAbs.

[0052] The term "single domain antibody" or "sdAb" refers to an antibody that contains three complementarity-determining regions (CDs). R). An sdAb refers to a single antigen-binding polypeptide having a corresponding CD It is possible for the R-containing polypeptide to bind to the antigen without pairing with it. Single domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are , herein referred to as "V H Some V H H is also known as a nanobody Camelid sdAbs are one of the smallest known antigen-binding antibody fragments. (e.g., Hamers-Casterman et al., Nature 3 63:446-8(1993);Greenberg et al.,Nature 3 74:168-73(1995); Hassanzadeh-Ghassabeh et al. al., Nanomedicine (Lond), 8: 1013-26 (2013) (See the basic V H H has the following structure from the N-terminus to the C-terminus: FR1-CD R1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 indicate framework regions 1 to 4, respectively, and CDR1 to CDR3 indicate complementarity-determining regions. Refers to 1 to 3.

[0053] An "isolated" antibody is one that has been identified and isolated from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is one that has been isolated and / or recovered. free from association with all other components from the production environment of the product. Contaminant components of its production environment, such as those causing toxic reactions, may be present in the antibody's research, diagnostic, or therapeutic use. These include enzymes, hormones, and other proteins. In a preferred embodiment, the solute may be a polypeptide or a non-proteinaceous solute. The antibody is (1) more than 95% by weight of the antibody, as determined, for example, by the Lowry method, and some In an embodiment, (1) a spinning cup sequenator is used to obtain a sieve containing more than 99% by weight of the sieve. sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence. or (3) non-reduced staining using Coomassie blue or preferably silver staining. or purified to homogeneity by SDS-PAGE under reducing conditions. The engineered antibody will not have at least one component of the antibody's natural environment present; However, in general, isolated polypeptides or The antibody is prepared by at least one purification step.

[0054] The "variable region" or "variable domain" of an antibody refers to the amino-terminal portion of a heavy or light chain of the antibody. The variable domains of the heavy and light chains are referred to as "V H " and "V L " These domains are generally the most variable parts of the antibody (those of the same class Heavy chain-only antibodies from Camelidae species contain the antigen-binding site (compared to other antibodies from other species). It has a single heavy chain variable region, which is referred to as "V H H". Therefore, V H H is V H It is a special type of

[0055] The term "variable" refers to the fact that certain segments of the variable domains are different in sequence from one another in antibodies. V domains mediate antigen binding and are responsible for determining the identity of a particular antibody. The variability, however, is uniform throughout the variable domains, defining their specificity for a particular antigen. Rather, it is distributed evenly across the variable domains of both the light and heavy chains. In the human genome, the variable domains are concentrated in three segments called hypervariable regions (HVRs). The more highly conserved parts of the heavy chain are called framework regions (FR). The variable domains of the α and β chains each connect a beta-sheet structure and in some cases , connected by three HVRs that form loops that form part of the beta-sheet structure The HVRs of each chain contain four FR regions that largely adopt a beta-sheet configuration. The HVRs from the other chain are held together in close proximity by the HVR domains, which together form the antibody's anti- Contributes to the formation of the primary binding site (Kabat et al., Sequences of Immunological Interest,Fifth Edition,Nat ional Institute of Health,Bethesda,Md.(1 991). The constant domains are not involved directly in binding the antibody to an antigen, but It exhibits various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity.

[0056] The term "monoclonal antibody" as used herein refers to a substantially homogenous antibody. It refers to antibodies obtained from a population of antibodies, i.e., the individual antibodies in the population are present in minute amounts. Possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amino acid substitution) that may occur Monoclonal antibodies are highly specific and target a single antigen. Different antibodies directed against different determinants (epitopes) are typically used. In contrast to polyclonal antibody preparations containing In addition to their specificity, monoclonal antibodies are also known for their ability to target specific determinants. It is synthesized by hybridoma culture and is free from contaminating immunoglobulins. The modifier "monoclonal" refers to a antibody derived from a substantially homogeneous population of antibodies. This indicates that the antibody is characterized as being capable of producing a specific antibody, and is interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present application should not be , hybridoma method (e.g., Kohler and Milstein, Nature e,256:495-97(1975);Hongo et al.,Hybridom a,14(3):253-260(1995), Harlow et al., Anti bodies:A Laboratory Manual,(Cold Spring Harbor Laboratory Press,2 nd ed.1988);Ham Merling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681(Elsevier,N .Y., 1981), recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567), see references), phage display technology (e.g., Clackson et al., Nature,352:624-628(1991);Marks et al.,J. Mol.Biol.222:581-597(1992);Sidhu et al., J.Mol.Biol.338(2):299-310(2004);Lee et a l.,J.Mol.Biol.340(5):1073-1093(2004);Fel louse,Proc.Natl.Acad.Sci.USA 101(34):124 67-12472(2004); and Lee et al., J. Immunol. Me Methods 284(1-2):119-132(2004)), and A portion of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence The technology for producing human antibodies or human-like antibodies in animals with all the above features has been developed (see, for example, WO199 8 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 10741, Jakobovits et al.,Proc.Natl.Acad. Sci.USA 90:2551(1993);Jakobovits et al., Nature 362:255-258(1993);Bruggemann et a I., Year in Immunol. 7:33 (1993); U.S. Patent No. 5,545 , No. 807, No. 5,545,806, No. 5,569,825, No. 5,625, Nos. 126, 5,633,425, and 5,661,016, Marks e t al.,Bio / Technology 10:779-783(1992);Lo nberg et al.,Nature 368:856-859(1994);Mo rrison,Nature 368:812-813(1994);Fishwild et al.,Nature Biotechnol.14:845-851(199 6);Neuberger,Nature Biotechnol.14:826(19 96); and Lonberg and Huszar, Intern. Rev. Im by various techniques, including (see, e.g., Mut. 13:65-93 (1995) can be made.

[0057] The term "naked antibody" refers to an antibody that is conjugated to a cytotoxic moiety or a radiolabel. This refers to antibodies that do not bind to the antibody.

[0058] The terms "full length antibody," "intact antibody," or "whole antibody" refer to antibody fragments. Used interchangeably to refer to an antibody in its substantially intact form, as opposed to a Specifically, it includes those having heavy and light chains that include a full-length four-chain antibody Fc region. Only antibodies have heavy chains (V H The constant domains include native sequence constant domains (e.g., H) and Fc regions. It may be a main (e.g., human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions. .

[0059] An "antibody fragment" is a portion of an intact antibody, preferably an antigen of the intact antibody. It contains a binding region and / or a variable region. Examples of antibody fragments include Fab, Fab ', F(ab')2, and Fv fragments; diabodies; linear antibodies (U.S. Patent No. 5, No. 641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; single-domain antibodies ( V H H, etc.), as well as multispecific antibodies formed from antibody fragments. Papain digestion of the body produces two identical antigen-binding fragments called "Fab" fragments. and the remaining "Fc" fragment, designated to reflect its ability to readily crystallize. The Fab fragment contains the entire L chain as well as the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1) Each Fab fragment Antibodies are monovalent with respect to antigen binding, i.e., they have a single antigen-binding site. Pepsin treatment yields a single large F(ab')2 fragment, which is The antibody is composed of two Fab fragments with the same antigen-binding activity, which are disulfide-bonded together. Fab' fragments are fragments of antibody fragments that are still capable of cross-linking antigens. C, including one or more cysteines from the C region H Several additional amino acids are added to the carboxy terminus of one domain. Fab'-SH fragments differ from Fab fragments by having additional residues. The cysteine ​​residue(s) of Fab' in the present specification have a free thiol group. F(ab')2 antibody fragments are originally F(ab')2 fragments with hinge cysteines between them. Other chemical modifications of antibody fragments have been reported. Coupling is also known.

[0060] The Fc fragment consists of the carboxyl groups of both heavy chains held together by disulfides. The effector functions of antibodies are determined by the sequences in the Fc region. Regions are also recognized by Fc receptors (FcRs) found on certain cell types.

[0061] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment contains one heavy chain variable region domain and one light chain variable region domain. The folding of these two domains results in a dimer of two domains tightly bound by non-covalent bonds. six amino acid residues that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. The hypervariable loops (three loops each from the H and L chains) are generated. A single variable domain (or HVR) specific for the antigen, but with lower affinity than the binding site. Even half of an Fv containing only three Fv fragments has the ability to recognize and bind to antigen. .

[0062] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a single Fv consisting of Fv fragments linked together to form a single polypeptide chain. V as a peptide chain H and V L It is an antibody fragment containing an antibody domain. For example, the sFv polypeptide is H and V L A polypeptide linker may be further provided between the domains. This allows the sFv to form the desired structure for antigen binding. For a review, see Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds., Springer-Verlag, New York. , pp. 269-315 (1994).

[0063] A "functional fragment" of an antibody as described herein includes a portion of an intact antibody. , which generally includes the antigen-binding or variable region of an intact antibody, or an FcR The Fc region of an antibody that retains or has modified FcR binding ability is included. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and antibody fragments. Examples of such antibodies include multispecific antibodies formed from antibodies.

[0064] The term "diabody" refers to a body that achieves inter-chain, but not intra-chain, V-domain pairing and thereby This results in a bivalent fragment, i.e., a fragment with two antigen-binding sites. V H and V L sFv with a short linker (approximately 5-10 residues) between the domains Small antibodies prepared by constructing fragments (see previous paragraph) Bispecific diabodies are fragments of two antibodies. H and V L Domain Heterodimers of two "crossover" sFv fragments present on different polypeptide chains Diabodies are described, for example, in European Patent No. 404,097 and International Publication No. WO93 / 11161, Hollinger et al., Proc. Natl. Acad. See Sci. USA 90:6444-6448 (1993).

[0065] As used herein, a monoclonal antibody refers to an antibody in which a portion of the heavy and / or light chain is specific to a particular species. corresponding in antibodies derived from or belonging to a particular antibody class or subclass The sequence is identical or homologous to the rest of the strand(s), while the rest of the strand(s) is / are from another species or are identical or identical to corresponding sequences in antibodies belonging to another antibody class or subclass. "Chimeric" antibodies (immunoglobulins), which are species specific, as well as fragments of such antibodies These compounds are optionally included in the present invention, provided that they exhibit the desired biological activity (see U.S. Pat. No. 4,811,226). 6,567;Morrison et al.,Proc.Natl.Acad.Sci USA, 81:6851-6855 (1984)). The antibody includes the PRIMATTZFD® antibody, wherein The antigen-binding region can be generated, for example, by immunizing macaques with the antigen of interest. As used herein, a "humanized antibody" is a "chimeric antibody." is used as a subset of

[0066] "Humanized" forms of non-human (e.g., camelid) antibodies are derived from non-human immunoglobulins. In some embodiments, a humanized antibody is a chimeric antibody that contains the minimal sequence of the recipient Residues from the HVRs (defined below) of the target polypeptide provide the desired specificity, affinity, and / or or competent non-human species such as mouse, rat, rabbit, or non-human primate (donor antigen human immunoglobulin (recipient antibody) in which residues from the HVRs of the In some cases, framework ("FR") residues of human immunoglobulins In addition, humanized antibodies are characterized by the fact that the amino acid residues of the target protein are replaced with corresponding non-human residues. These modifications may include residues that are not found in either the host or donor antibody. Generally, humanized antibodies have at least one typical Typically, it will contain substantially all of the two variable domains, where the hypervariable loops all or substantially all of the FR regions correspond to those of a non-human immunoglobulin sequence, or substantially all correspond to those of human immunoglobulin sequences, but the FR regions contain One or more individual FR residue substitutions that improve antibody performance, such as affinity, isomerization, or immunogenicity. The number of these amino acid substitutions in the FR is typically 6 in the H chain. The humanized antibody may also optionally be a humanized antibody having an immunoglobulin constant of 0.1 or less. At least a portion of the common region (Fc), typically at least a portion of a human immunoglobulin For further details, see, for example, Jones et al., Nature 321:522-525(1986);Riechmann et al.,Natur e 332:323-329 (1988); and Presta, Curr. Op. Str. See, e.g., Vasw. ani and Hamilton,Ann.Allergy,Asthma & Im munol.1:105-115(1998);Harris,Biochem.Soc .Transactions 23:1035-1038(1995);Hurle a nd Gross,Curr.Op.Biotech.5:428-433(1994) and U.S. Patent Nos. 6,982,321 and 7,087,409. stomach.

[0067] A "human antibody" is an antibody that contains an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human. and / or any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody includes non-human antigen-binding residues. Human antibodies are specifically excluded from the scope of human antibodies, including humanized antibodies derived from phage display libraries. It can be produced using a variety of techniques known in the art. Winter, J.Mol.Biol.,227:381(1991);Marks e t al., J. Mol. Biol., 222:581 (1991). Cole et al., Monoclonal Antibodies and Cancer The rapy,Alan R.Liss,p.77(1985);Boerner et a I., J. Immunol., 147(1):86-95(1991) are also available for the preparation of human monoclonal antibodies. van de Winkel,Curr.Opin.Pharmacol.,5:36 See also, 8-74 (2001). Human antibodies are produced in response to antigen challenge. Transgenics that are modified to produce a gene that is inactivated at its endogenous locus The antigen can be prepared by administering the antigen to a mouse, such as an immunized xenogeneic mouse (e.g., , and XENOMOUSE™ technology, see U.S. Pat. No. 6,075,181 and See, e.g., Li et al., Proc. Natl. Acad. Sci. Also see tl.Acad.Sci.USA103:3557-3562(2006) stomach.

[0068] As used herein, the terms "hypervariable region," "HVR," or "HV" are antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. The term "HVR" refers to the region of a domain. Generally, single domain antibodies have three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR4) HVR3 exhibits the highest diversity of the three HVRs and is the most suitable for antibody synthesis. For example, Hamers-Cas Terman et al.,Nature 363:446-448(1993);S heriff et al.,Nature Struct.Biol.3:733-7 36(1996).

[0069] The term "complementarity determining region" or "CDR" is defined according to the Kabat system. It is used to refer to hypervariable regions such as those in Kabat et al., Sequen ces of Proteins of Immunological Interes t,5th Ed.Public Health Service,National Institutes of Health, Bethesda, Md. (1991) Please refer to.

[0070] Several HVR delineations are used herein and are encompassed herein. Kabat complement The sex-determining regions (CDRs) are the most commonly used because of their sequence variability. (Kabat et al., Sequences of Proteins of I mmunological Interest,5th Ed.Public Heal th Service,National Institutes of Health (See, e.g., Bethesda, Md. (1991)). Chothia, instead The positions of the structural loops are shown in Fig. 1 (Chothia and Lesk, J. Mol. Biol. l.196:901-917(1987)). AbM HVR is similar to Kabat HVR. Chothia structural loops, and Oxford Molecular Used by AbM antibody modeling software. "Contact" HVRs are available Based on analysis of the complex crystal structure, the residues of each of these HVRs are listed in Table 1 below. . [Table 1]

[0071] HVRs may include the following "extended HVRs": L In 24~36 or 24~ 34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and V H In the case of 26-35 (H1), 50-65 or 49-65 ( H2), and 93-102, 94-102, or 95-102 (H3). For each of these definitions, the residues are defined as follows according to Kabat et al. (see above): They are numbered.

[0072] Single domain antibodies (V H The amino acid residues of the hydroxyl groups (H, etc.) are determined by Riechmann and Mu yldermans,J.Immunol.Methods 2000 Jun.23; 240(1-2):185-195, V from the Camelidae family H Suitable for H domain As used in Kabat et al. ("Sequence of protein ns of immunological interest”, US Public Health Services,NIH Bethesda,Md.,Publica tion No.91) H A common numbering scheme for domains is According to this numbering, V H FR1 of H is the amino acid sequence 1-30 Contains acid residues, V H CDR1 of H contains amino acid residues 31 to 35, and CDR1 of V H HFR 2 contains amino acid residues 36 to 49, and V H CDR2 of H is the amino acid sequence at positions 50-65. Contains acid residues, V H FR3 of H contains amino acid residues 66 to 94, and V H H CDR 3 contains amino acid residues 95 to 102, and V H FR4 of H is 103-113 In this regard, it is well known in the art that V H About the domain Te and V H For the H domain, the total number of amino acid residues in each of the CDRs can vary. may not correspond to the total number of amino acid residues indicated by Kabat numbering ( That is, if one or more positions according to the Kabat numbering are unoccupied in the actual sequence, or the actual sequence is greater than the number allowed by the Kabat numbering. It should be noted that the amino acid residues may be present.

[0073] "Variable domain residue numbering as in Kabat" or "Kabat The phrase "simple amino acid position numbering" and variations thereof are used in accordance with Kabat et al. (supra). used in the heavy chain variable domain or light chain variable domain in the construction of antibodies in This numbering system is used to identify the actual linear amino acid sequence. The smaller fragments correspond to truncations of, or insertions into, the FRs or HVRs of the variable domain. For example, the heavy chain variable domain may contain residues of H2 or additional amino acids. A single amino acid insertion after residue 52 (residue 52a according to Kabat) and the heavy chain FR residues Residues inserted after group 82 (e.g., residues 82a, 82b, and 82a according to Kabat) The Kabat numbering of residues may be used to determine the sequence of the antibody for a given antibody. determined by matching the homologous regions with the "standard" Kabat-numbered sequence. It can be done.

[0074] Unless otherwise indicated herein, the numbering of residues within an immunoglobulin heavy chain is as follows: The EU indicators are as in Kabat et al. "EU index" refers to the residue numbering of the human IgG1 EU antibody.

[0075] "Framework" or "FR" residues are those residues other than, possibly, HVR residues as herein defined. These are variable domain residues.

[0076] A "human consensus framework" or "acceptor human framework" is Human Immunoglobulin V L or V H The most common occurrence in framework sequence selection is It is a framework that represents the amino acid residues that make up the human immunoglobulin V. L or V H The selection of sequences is from a subgroup of variable domain sequences. Generally, a subgroup of sequences The group is based on Kabat et al., Sequences of Proteins f Immunological Interest,5 thEd.Public H health service,National Institutes of Hea Subgroups such as those in the University of Illinois, Bethesda, Md. (1991). So, V L The subgroup includes those related to Kabat et al. Subgroups kappa I, kappa II, kappa III, or kappa IV, as in Furthermore, for VH, subgroups may be selected as in Kabat et al. It may be subgroup I, subgroup II, or subgroup III. In the human consensus framework, certain residues, e.g., the donor framework sequence, The human framework sequences were identified by aligning the sequence with a collection of different human framework sequences. Residues may be selected based on their homology to the donor framework, such as those described above. It may be derived from a human immunoglobulin framework or a human consensus framework. The acceptor human framework "derived from" the framework contains the same amino acid sequence. or it may contain pre-existing amino acid sequence changes. In this case, the number of existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less. The following are: 5 or less, 4 or less, 3 or less, or 2 or less.

[0077] For example, an "amino acid modification" at a specified position in the Fc region refers to the substitution of the specified residue. substitution or deletion, or insertion of at least one amino acid residue adjacent to the specified residue An insertion "adjacent" to a specified residue means an insertion within one or two residues of that residue. The insertion may be N-terminal or C-terminal to the specified residue. A preferred amino acid modification is a substitution.

[0078] An "affinity matured" antibody has one or more changes in one or more of its HVRs, and these changes but a decrease in the affinity of the antibody for the antigen compared to a parent antibody that does not have those alterations(s). In some embodiments, affinity matured antibodies provide improved antibody binding to a target antigen. Affinity matured antibodies have nanomolar or even picomolar affinities for antibodies against IgG. It is produced in the brain by known procedures. See, e.g., Marks et al., Bio / Te Technology 10:779-783(1992) H and V L Domain Shaft The authors describe affinity maturation by randomizing HVR and / or framework residues. Dam mutagenesis is described, for example, in Barbas et al. Proc Nat. Acad. S ci.USA 91:3809-3813(1994);Schier et al.G ene 169:147-155(1995);Yelton et al.J.Imm unol.155:1994-2004(1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992) There are.

[0079] As used herein, "specifically bind," "specifically recognize," or "specifically recognize" The term "specific for" refers to the binding of a target to an antigen-binding protein (e.g., CAR or sdAb). It refers to measurable and reproducible interactions, such as binding between molecules, including biomolecules. Determine the presence of the target in the presence of a heterogeneous population of antigens. For example, Antigen binding proteins (such as CARs or sdAbs) that specifically bind to the target molecule can be used to target other targets. binds with higher affinity, avidity, more readily, and / or with longer duration than It is an antigen-binding protein (such as a CAR or sdAb) that binds to this target. In some embodiments, the antigen binding protein (such as a CAR or sdAb) binds to an unrelated target. The degree to which the antibody binds to the antigen, as measured, for example, by radioimmunoassay (RIA), is In some embodiments, the protein (such as a CAR or sdAb) is less than about 10%. The antigen-binding protein (e.g., CAR or sdAb) that specifically binds to the target is at least 1 μM. Dissociation constant (K In some embodiments, the antigen binding protein (such as a CAR or sdAb) ) specifically binds to epitopes on proteins that are conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. Do not.

[0080] The term "specificity" refers to the ability of an antigen-binding protein (CA) to bind to a particular epitope of an antigen. This refers to the selective recognition of specific antibodies (e.g., sdAb or sdAb). For example, natural antibodies are monospecific. The term "multispecific" as used herein refers to an antigen binding protein (CAR or or sdAb, etc.) have two or more antigen-binding sites, at least two of which are different. "Bispecific" refers to binding to the same antigen or to different epitopes of the same antigen. As used herein, an antigen binding protein (such as a CAR or sdAb) is a protein that binds two The term "monospecific" CAR refers to CARs having different antigen binding specificities. As used herein, one or more binding sites, each of which binds to the same epitope on the same antigen. 1 shows an antigen binding protein (such as a CAR or sdAb) having the following structure:

[0081] The term "valent" as used herein refers to the amount of an antigen binding protein (CAR or indicates the presence of a specified number of binding sites in a given antibody, e.g., a natural antibody, or Full-length antibodies have two binding sites and are bivalent. Thus, "trivalent," "tetravalent," " The terms "pentavalent" and "hexavalent" refer to the antigen binding protein (such as a CAR or sdAb). 2 binding sites, 3 binding sites, 4 binding sites, and 5 binding sites, respectively. , and indicates the presence of six binding sites.

[0082] The "effector functions" of an antibody are determined by the Fc region (native sequence Fc region or amino acid The biological activity attributable to the sequence variant Fc region varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc Receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g. "Reduced or minimized" includes downregulation of B cell receptors; and B cell activation. The modified antibody effector function is at least 50% (or more) of that of the wild-type or unmodified antibody. are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, and 99% reduction in antibody effector function. In a preferred embodiment, complement fixation, complement binding, The antibody effector functions of antibody-dependent cellular cytotoxicity and antibody-dependent cellular cytotoxicity are affected. In some embodiments, the effector function is determined by a variation in the constant region that excludes glycosylation. In one embodiment, the effector is eliminated through mutation, e.g., "effectorless mutation." The less mutation is C H N297A or DANA mutation in two regions (D265A + N297 A). Shields et al., J. Biol. Chem. 276(9): 6591-6604(2001). Alternatively, reduced or eliminated effector function Additional mutations that result in leukemia include K322A and L234A / L235A (LALA). Alternatively, the effector functions can be expressed in non-glycosylated host cells (e.g., E. coli), or are ineffective or too weak in promoting effector function. expression in host cells that result in altered glycosylation patterns, etc. can be reduced or eliminated through production techniques (e.g., Shinkawa et al. .,J.Biol.Chem.278(5):3466-3473(2003).

[0083] "Antibody-dependent cell-mediated cytotoxicity" or ADCC is a method for detecting the activity of certain cytotoxic cells (e.g., Fc receptors present on cells such as natural killer (NK) cells, neutrophils, and macrophages Secreted Ig bound to the FcR allows these cytotoxic effector cells to target antigen-bearing targets. It specifically binds to cells and can then kill the target cells using a cytotoxin. This refers to a form of cytotoxicity that targets the body's immune system. Antibodies are "armed" with cytotoxic cells that target the body's immune system by this mechanism. Necessary for cell killing. NK cells are the primary cells for mediating ADCC. While human leukocytes express only FcγRIII, monocytes express FcγRI, FcγRII, and FcγRIII. γRIII. Fc expression on hematopoietic cells was reported by Ravetch and Kinet , Annu. Rev. Immunol. 9:457-92 (1991), Table 3, p. 464 To assess the ADCC activity of a molecule of interest, the methods described in U.S. Pat. In vitro ADC assays such as those described in US Pat. Nos. 5,821,337 and 5,821,362. Useful effector cells for such assays include terminal These include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be demonstrated in vivo, e.g., by immunohistochemistry using Clynes et al., PNAS USA 95:652-656 (1998), etc. The present invention may also be evaluated in animal models.

[0084] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain. The term "Fc region" is used herein to refer to a specific Fc region, and includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of a human IgG heavy chain can vary, the human IgG heavy chain Fc region typically begins at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) For example, during the production or purification of the antibody, or by recombinantly manipulating the nucleic acid encoding the heavy chain of the antibody. Thus, compositions of intact antibodies may contain the entire K447 residue. an antibody population with the K447 residue removed, an antibody population without the K447 residue removed, and an antibody population with the K447 residue removed. The antibody population may include a mixture of antibodies that have and do not have the antibodies described herein. Native sequence Fc regions suitable for use in antibodies include human IgG1, IgG2 (Ig IgG1A, IgG2B), IgG3, and IgG4.

[0085] "Binding affinity" generally refers to the affinity of a single binding site of a molecule (e.g., an antibody or CAR) to its It refers to the strength of the total non-covalent interactions between a binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the affinity of a member of a binding pair (e.g., specific interactions that reflect a 1:1 interaction between the antibody and antigen, or the CAR and antigen. The affinity of a molecule X for its partner Y is generally expressed as the dissociation constant ( Affinity can be expressed as a function of the affinity of the target molecule, or Kd. ... Low affinity antibodies generally bind antigens slowly and readily. High affinity antibodies generally bind antigens faster and remain bound for longer, whereas high affinity antibodies tend to dissociate rapidly. Various methods for measuring binding affinity are known in the art. Any of these may be used for the purposes of this application. Specific illustrative and exemplary embodiments for determining the same are described below.

[0086] "Blocking" or "antagonist" antibodies inhibit the biological activity of the antigen to which they bind. In some embodiments, blocking or antagonist antibodies The body substantially or completely inhibits the biological activity of the antigen.

[0087] An "agonist" or activating antibody enhances or inhibits signaling by the antigen to which it binds. In some embodiments, agonist antibodies inhibit or initiate the activity of natural ligands. Triggers or activates signal transduction in the absence of

[0088] With respect to peptide, polypeptide, or antibody sequences, "percent amino acid sequence identity" is used. "(%)" and "homology" refer to alignment of sequences to achieve the maximum percent sequence identity. and, after introducing gaps if necessary, any conservative residues as part of the sequence identity. The amino acid residues in a particular peptide or polypeptide sequence, without taking substitutions into account, are identical to Amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical. Matching for purposes of determining percentages can be accomplished by a variety of methods within the skill of the art. For example, BLAST, BLAST-2, ALIGN, or MEGALIGN (商標 ) Use publicly available computer software such as (DNASTAR) software. Those skilled in the art will find that the best possible match is achieved over the entire length of the sequences being compared. Determine appropriate parameters for measuring the match, including any algorithms required for It is possible.

[0089] As used herein, a "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that binds to an immune receptor such as a T cell. Genetically engineered vectors that can be used to graft one or more antigen specificities onto effector cells. Some CARs are called "artificial T cell receptors," "chimeric T cell receptors," ", or "chimeric immune receptor." In some embodiments, a CAR comprises one or more antigens (such as tumor antigens) of T cell and / or other receptors, a transmembrane domain, and an extracellular antigen-binding domain specific to an intracellular signaling domain. "T" refers to a T cell expressing a CAR.

[0090] An "isolated" nucleic acid molecule encoding a CAR or sdAb described herein is one that and identifying and isolating the nucleic acid from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, the isolated nucleic acid is free of all associated nucleic acid molecules in the production environment. Isolated nucleic acids encoding the polypeptides and antibodies herein, free from association with components. A molecule is in a form other than the form or setting in which it is found in nature. The nucleic acid molecules encoded by the polypeptides and antibodies of the present invention are those naturally occurring in cells. It is distinct from acid.

[0091] The term "control sequence" refers to an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoter sequences, promoter regions, and promoter sequences. The promoter sequence may include a promoter, an optional operator sequence, and a ribosome binding site. They are known to use promoters, polyadenylation signals, and enhancers.

[0092] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, presequence or secretory leader DNA is involved in the secretion of a polypeptide. When expressed as a preprotein, it is operably linked to the DNA of the polypeptide. or a promoter or enhancer if it affects the transcription of a coding sequence. , operably linked to the sequence, or a ribosome binding site that When positioned to facilitate transcription, it is operably linked to a coding sequence. "Operably linked" means that the DNA sequences being linked are contiguous and secreted. In the case of a leader, it means that it is continuous and in the leading phase. Therefore, enhancers do not have to be contiguous. Linkage can be achieved by ligation at convenient restriction sites. If no such site exists, this can be achieved by a synthetic oligonucleotide. Adapters or linkers are used according to conventional practice.

[0093] As used herein, the term "vector" refers to a nucleic acid molecule to which another nucleic acid molecule is linked. The term refers to a nucleic acid molecule that can propagate itself as a self-replicating nucleic acid structure. and vectors integrated into the genome of the host cell into which they are introduced. Such vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0094] As used herein, the term "autologous" refers to a gene that is subsequently reintroduced into an individual. "Material" is intended to refer to any material derived from the same individual.

[0095] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0096] As used herein, "transfected" or "transformed" or " The term "transduced" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. "Transfected" or "transformed" or "transduced" cells are cells that have been infected with an exogenous nucleus. A cell that has been transfected, transformed, or transduced with an acid. The cell is a primary It includes the subject cell and its progeny.

[0097] As used herein, the terms "cell," "cell line," and "cell culture" refer to , and are used interchangeably, and all such designations include progeny. Thus, "transfector" refers to a "Incubate" and "transfected cells" refer to the primary subject cell and its subsequent transfers regardless of the number of transfers. All progeny may vary in DNA content due to deliberate or accidental mutations. It is understood that the cells may not be strictly identical in terms of the degree of their transformation. The mutant progeny have the same function or biological activity as screened for. do.

[0098] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably. refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. "Transformants" and "transformed cells" include the primary transformed cells and successively transformed cells. It includes progeny derived therefrom regardless of generation. Progeny are completely identical in nucleic acid content to the parent cell. The resulting transformed cells are not identical to the original but may contain mutations. or mutant progeny that have the same function or biological activity as the selected one are included herein. It can be enjoyed.

[0099] As used herein, "treatment" or "treating" refers to a condition that results in beneficial effects, including clinical results. or an approach to achieving a desired result. Desired clinical outcomes include: alleviation of one or more symptoms caused by the disease, a reduction in the severity of the disease, progression, disease stabilization (e.g., prevention or delay of disease progression), disease spread (e.g., metastasis) ) prevention or delay of disease recurrence, delay or slow progression of disease, symptom improvement in the condition, providing remission (partial or total) of the disease, or providing one or more other therapeutic agents necessary for the treatment of the disease Reduced drug dose, delayed disease progression, improved quality of life, and / or prolonged survival The reduction of the pathological consequences of cancer, including, but not limited to, one or more of the following, is also considered a "treatment." The methods of the present application contemplate any one or more of these therapeutic modalities. do.

[0100] As used herein, an "individual" or "subject" refers to a human, bovine, equine, feline, Refers to a mammal, including but not limited to a dog, rodent, or primate. In an embodiment, the individual is a human.

[0101] As used herein, the term "effective amount" refers to an amount effective to treat a particular disorder, condition, or disease. to treat a condition, e.g., to improve, alleviate, relieve, and / or delay one or more of its symptoms. an amount of an agent sufficient to induce the immune response, e.g., a single domain antibody, an engineered immune effector cell, or the amount of pharmaceutical composition thereof. With respect to cancer, an effective amount refers to an amount that causes tumor shrinkage, and / or slow the rate of tumor growth (e.g., suppress tumor growth) or other In some embodiments, the amount of the compound is sufficient to inhibit or retard unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the onset of the disease. An effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more doses. An effective amount of the drug or composition can be administered to (i) reduce the number of cancer cells, and (ii) prevent the progression of the tumor. (iii) prevent, delay, and to some extent slow cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent) tumor metastasis; (v) inhibit tumor growth; and (vi) prevent tumor onset and / or recurrence. (vii) prevent or delay, and / or alleviate to some extent, one or more symptoms associated with cancer; The degree of damage can be reduced.

[0102] "Adjuvant setting" refers to an individual who has a history of cancer and who has generally (but not necessarily) undergone surgery. Therapies including, but not limited to, (e.g., resective surgery), radiation therapy, and chemotherapy However, due to a solid cancer history, these individuals , are considered at risk of developing the disease. Administration refers to the subsequent treatment modality. The degree of risk (e.g., individual in an adjuvant setting) When a body is considered "high risk" or "low risk" depends on several factors, most commonly The specific treatment will depend on the extent of the disease when first treated.

[0103] "Neoadjuvant setting" refers to the clinical setting in which the method is administered before primary / causal therapy .

[0104] As used herein, "delaying" the onset of cancer means suspending or preventing the onset of the disease. This means to impair, delay, retard, stabilize, and / or postpone. The duration may vary depending on the medical history and / or individual being treated. As such, a sufficient or significant delay can be such that the individual does not, in effect, develop the disease. A method that "delays" the onset of cancer compared to not using the method can encompass prevention. to reduce the likelihood of disease onset within a given time frame and / or to prevent disease onset within a given time frame. Such a comparison is typically performed on a statistically significant number of individuals. Cancer incidence is based on clinical studies using computerized tomography (CAT scan), magnetic resonance imaging (MRI), and including magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy The onset of the disease may be detectable using standard methods, including but not limited to, It also refers to possible cancer progression, including onset, recurrence, and development.

[0105] The term "pharmaceutical formulation" refers to a form in which the biological activity of an active ingredient is made effective, and a preparation that does not contain additional components that are unacceptably toxic to the subject to whom the formulation is administered. Such a preparation is sterile. A "sterile" preparation is one that is aseptic or free of all viable microorganisms. Does not include living organisms and their spores.

[0106] As used herein, a "carrier" refers to a substance that is exposed to the It includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of carriers include buffers such as phosphate, citric acid, and other organic acids; ascorbic acid and Antioxidants including methionine; Preservatives (octadecyldimethylbenzylammonium chloride) ;Hexamethonium chloride;Benzalkonium chloride, Benzethonium chloride;Phenol, alkylparabens, e.g., methyl or propyl parabens; Laven; Catechol; Resorcinol; Cyclohexanol; 3-Pentanol; and m -cresols, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., blood serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinyl Pyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or Dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol , trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, e.g., TWEEN™, polyethylene glycol (PEG), and PLURONICS™ Examples include polyethylene glycol (PEG), or polyethylene glycol (PEG).

[0107] The intended "diluent" is defined herein as being pharmaceutically acceptable (suitable for administration to humans). safe and non-toxic), for the preparation of liquid formulations, e.g., for formulations that are lyophilized and then reconstituted. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or Dextromethorphan In an alternative embodiment, the diluent is an aqueous solution of salt and / or or a buffer.

[0108] "Preservative" refers to a compound that may be added to the formulations herein to reduce bacterial activity. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations. Examples of possible preservatives include, for example, octadecyldimethylbenzylammonium chloride. Benzalkonium chloride (with long alkyl chains) A mixture of alkylbenzyldimethylammonium chlorides (a compound of alkylbenzyldimethylammonium chloride) and benzethonium chloride. Other types of preservatives include aromatic alcohols, e.g. , phenol, butyl, and benzyl alcohol, alkyl parabens, e.g., methyl or or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentaerythritol The most preferred preservatives herein are benzyl alcohol, methyl alcohol, and m-cresol. It is benzyl alcohol.

[0109] A "stable" formulation is one in which the protein maintains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available. Peptide and Protein Drug D elivery,247-301,Vincent Lee Ed.,Marcel D ekker, Inc., New York, NY, Pubs. (1991) and Jo nes, A.Adv.Drug Delivery Rev.10:29-90(199 3) Stability is measured at a selected temperature over a selected period of time. For rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month. When the formulation is stored at 2-8°C, the stability is generally measured at 30°C. It should be stable for at least one month at 40°C or 2-8°C. It should be stable for at least 2 years. When the formulation is stored at 30°C, The agent should be stable for at least 2 years at 30°C and / or at least 40°C. Both should be stable for 6 months. For example, the degree of aggregation during storage may be a factor in determining protein stability. Therefore, a "stable" formulation would contain approximately 10% of the protein. Less than about 5%, preferably less than about 5%, may be present as aggregates in the formulation. Morphology can determine any increase in aggregate formation during storage of the formulation.

[0110] A "reconstituted" formulation is a lyophilized form of a protein that has been reconstituted in a diluent such that the protein is dispersed throughout. It is prepared by dissolving a protein or antibody preparation. The formulation is suitable for administration (e.g., subcutaneous administration) to a patient to be treated with the protein of interest. and in some embodiments of the present application may be suitable for parenteral or intravenous administration. do.

[0111] An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Generally, it has an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" refers to the osmotic pressure of human blood. Similarly, the term "hypertonic" describes a formulation that has an osmolality lower than that of a human It is used to describe a formulation that has an osmotic pressure higher than that of blood. For example, the vapor pressure can be measured using a vapor pressure or ice osmometer. or hypertonic as a result of the addition of buffer.

[0112] It should be understood that the embodiments of the present application described herein "consist of" and / or "essentially consist of" embodiments. It is understood that this includes "becoming a target."

[0113] Reference herein to "about" a value or parameter refers to that value or parameter itself. For example, a statement referring to "about X" includes (and describes) variations that are directed to Contains the description "X".

[0114] As used herein, a reference to a value or parameter "not being" generally refers to , meaning and describing "other" than a certain value or parameter. For example, treating cancer type X. The method is not used to treat cancer types other than X. This means that

[0115] As used herein, the term "about XY" has the same meaning as "about X to about Y." .

[0116] As used in this specification and the appended claims, the singular forms "a," "or," and "a" are used interchangeably. "The" includes plural referents unless the context clearly dictates otherwise.

[0117] II. Single Domain Antibodies In one aspect, the present application relates to single domain antibodies, antigen-binding fragments thereof, and single domain antibodies. An exemplary single domain antibody is provided. The domain antibodies are listed in Table 2 below. [Table 2-1] [Table 2-2]

[0118] Anti-CD19 single domain antibody In one aspect, the present application provides an isolated single antibody that specifically binds to CD19, such as human CD19. In some embodiments, the anti-CD19 single domain antibody is a CD19 single domain antibody. In some embodiments, the anti-CD19 single domain antibody modulates anti-CD19 activity. It is an antagonist antibody.

[0119] In some embodiments, one, two, or all three of the amino acid sequences of SEQ ID NO: 76 In some embodiments, an anti-CD19 single domain antibody is provided, comprising the CDRs of: The CD19 single domain antibody is a camelid. In some embodiments, the anti-CD19 single domain antibody is a In some embodiments, the anti-CD19 single domain antibody is humanized. The host may also contain an acceptor human framework, such as a human immunoglobulin framework or or the Human Consensus Framework.

[0120] In some embodiments, the CDR1 comprises: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 3; DR3, at least one, at least two, or all three CDRs selected from Anti-CD19 single domain antibodies are provided. In some embodiments, the anti-CD19 single domain In some embodiments, the main antibody is a camelid. In some embodiments, the anti-CD19 single domain antibody is humanized. human frameworks, e.g., human immunoglobulin frameworks or human concepts Includes the Consus framework.

[0121] In some embodiments, (a) a nucleic acid sequence that is at least about 85%, 80%, or 90% identical to the amino acid sequence of SEQ ID NO: 1; 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% sequence identity. (b) a CDR1 that has at least about 85%, 86%, or 87% of the amino acid sequence of SEQ ID NO:2; ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% , 98%, 99%, or 100% sequence identity to CDR2 and (c) at least about 85%, 86%, 87%, 88% identical to the amino acid sequence of SEQ ID NO: 3. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% sequence identity to a CDR3; Anti-CD19 single domain antibodies comprising three CDRs are provided. In some embodiments, In some embodiments, the anti-CD19 single domain antibody is a camelid. In some embodiments, the antibody is at least about 85%, 86%, 87%, or 90% humanized. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identity to the reference sequence. Antibody containing the sequence but containing substitutions (e.g., conservative substitutions), insertions, or deletions. The CD19 single domain antibody retains the ability to bind to CD19. In some embodiments, the anti-CD19 antibody is an affinity matured antibody. In some embodiments, the anti-CD19 single domain antibody is a camelid. The antibody is humanized. In some embodiments, the anti-CD19 single domain antibody is Sceptor human frameworks, e.g., human immunoglobulin frameworks or human co- Includes consensus framework.

[0122] In some embodiments, the CDR1 comprises: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 3; Anti-CD19 single domain antibodies are provided that contain three CDRs, including DR3. In some embodiments, the anti-CD19 single domain antibody is a camelid. In some embodiments, the anti-CD19 antibody is humanized. The base antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin framework. The framework includes a human consensus framework.

[0123] In some embodiments, the amino acid sequence of SEQ ID NO: 76 is at least about 85%, 86%, or ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, 99%, or 100% sequence identity. V H Anti-CD19 single domain antibodies comprising an H domain are provided. is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Any one of the following: 93%, 94%, 95%, 96%, 97%, 98%, or 99% V has the identity of H The H sequence may contain substitutions (e.g., conservative substitutions), insertions, or or a deletion thereof, but containing the sequence thereof, binds to CD19. In some embodiments, a total of 1 to 10 amino acids are selected from the group consisting of SEQ ID NO: 76 amino acid substitutions, insertions, and / or deletions in the sequence. In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., Optionally, the anti-CD19 single domain antibody may include post-translational modifications of its sequence. In some embodiments, the anti-CD19 single domain antibody comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-CD19 antibody is humanized. In some embodiments, the anti-CD19 single domain antibody is an acceptor human frame. framework, e.g., human immunoglobulin framework or human consensus framework Includes work.

[0124] In some embodiments, a V has the amino acid sequence of SEQ ID NO: 76. H Contains the H domain Anti-CD19 single domain antibodies are provided. In some embodiments, the antibody of SEQ ID NO: 76 A polypeptide comprising the amino acid sequence is provided.

[0125] In some embodiments, functional epitopes are identified by combinatorial alanine scanning In this process, the sequence can be mapped by combinatorial alanine scanning. Using a cloning strategy, we identified the CD19 protein required for interaction with anti-CD19 single domain antibodies. In some embodiments, an epitope can identify an amino acid in a protein. Using the crystal structure of an anti-CD19 single domain antibody in the endothelial conformation and bound to CD19, In some embodiments, the present application provides the Specific for the same epitope as any one of the provided anti-CD19 single domain antibodies For example, in some embodiments, an antibody that binds to the amino acid sequence of SEQ ID NO: 76 is provided. Antibodies are provided that bind to the same epitope as an anti-CD19 single domain antibody comprising the sequence:

[0126] In some embodiments, the present application provides a method for the production of anti-CD19 single domain antibodies described herein. an anti-CD19 antibody that specifically binds to CD19 in competition with any one of the above, or In some embodiments, competitive binding is measured by ELISA. For example, in some embodiments, the amino acid sequence of SEQ ID NO: 76 can be determined using an assay. Antibody specifically binding to CD19 in competition with anti-CD19 single domain antibody containing the amino acid sequence The body is provided.

[0127] In some embodiments, any of the anti-CD19 single domain antibodies described above Anti-CD19 antibodies or antigen binding proteins comprising one or more of the following are provided: In certain embodiments, anti-CD19 antibodies may be monoclonal, including camelid, chimeric, humanized, or human antibodies. In some embodiments, the anti-CD19 antibody is an antibody fragment, e.g., a clonal antibody. For example, V H In some embodiments, the anti-CD19 antibody is an IgG H fragment. full-length heavy chains containing the Fc region of any antibody class or isotype, such as IgG1 or IgG4 In some embodiments, the Fc region is an antibody with reduced or minimized effect. It has a target function.

[0128] In some embodiments, the anti-CD19 antibody (anti- CD19 single domain antibodies) or antigen-binding proteins are characterized by the characteristics of the antibodies listed below. Any of the features described in sections 1 to 7 may be incorporated alone or in combination.

[0129] In some embodiments, the anti-CD19 antibodies described above (anti-CD19 single domain antibodies) Isolated nucleic acids encoding any one of the following (e.g., nucleotide sequences): In one embodiment, an isolated nucleic acid encoding an anti-CD19 single domain antibody is provided, The acid has at least about 85%, 86%, 87%, 88%, 89%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 101. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% sequence identity. In embodiments, an isolated nucleic acid is provided comprising the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, vectors (e.g., expression vectors) containing such nucleic acids are provided. In some embodiments, a host cell containing such nucleic acid is provided.

[0013] A method for producing an anti-CD19 antibody is provided, the method comprising: Host cells containing nucleic acid encoding the antibody are cultured under conditions suitable for expression of the anti-CD19 antibody. and optionally recovering the anti-CD19 antibody from the host cells (or host cell culture medium). Includes toto.

[0130] Anti-CD20 single domain antibody In one aspect, the present application provides an isolated single antibody that specifically binds to CD20, such as human CD20. In some embodiments, the anti-CD20 single domain antibody is In some embodiments, the anti-CD20 single domain antibody modulates anti-CD20 activity. It is an antagonist antibody.

[0131] In some embodiments, one, two, or all three of the amino acid sequences of SEQ ID NO: 77 In some embodiments, an anti-CD20 single domain antibody is provided, comprising the CDRs of The CD20 single domain antibody is a camelid. In some embodiments, the anti-CD20 single domain antibody is a In some embodiments, the anti-CD20 single domain antibody is humanized. The host may also contain an acceptor human framework, such as a human immunoglobulin framework or or the Human Consensus Framework.

[0132] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (d) CDR3 comprising the amino acid sequence of SEQ ID NO: 6. DR3, at least one, at least two, or all three CDRs selected from Anti-CD20 single domain antibodies are provided. In some embodiments, the anti-CD20 single domain In some embodiments, the main antibody is a camelid. In some embodiments, the anti-CD20 single domain antibody is humanized. human frameworks, e.g., human immunoglobulin frameworks or human concepts Includes the Consus framework.

[0133] In some embodiments, (a) a nucleic acid sequence that is at least about 85%, ... 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% sequence identity. (b) a CDR1 that has at least about 85%, 86%, or 87% of the amino acid sequence of SEQ ID NO: 5; ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% , 98%, 99%, or 100% sequence identity to CDR2 and (c) a sequence identical to the amino acid sequence of SEQ ID NO: 6, with a sequence identical to at least about 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% sequence identity to a CDR3; Anti-CD20 single domain antibodies comprising three CDRs are provided. In some embodiments, the anti-CD20 single domain antibody is a camelid. In some embodiments, the antibody is at least about 85%, 86%, 87%, or 90% humanized. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identity to the reference sequence. Antibody containing the sequence but containing substitutions (e.g., conservative substitutions), insertions, or deletions. The CD20 single domain antibody retains the ability to bind to CD20. In some embodiments, the anti-CD20 single domain antibody is an affinity matured antibody. The anti-CD20 single domain antibody is a camelid. In some embodiments, the anti-CD20 The single domain antibody is humanized. In some embodiments, the anti-CD20 single domain antibody The antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin framework. or human consensus framework.

[0134] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (d) CDR3 comprising the amino acid sequence of SEQ ID NO: 6. Anti-CD20 single domain antibodies comprising three CDRs, including DR3, are provided. In some embodiments, the anti-CD20 single domain antibody is a camelid. In some embodiments, the anti-CD20 single domain antibody is humanized. 20 Single domain antibodies are constructed using acceptor human frameworks, e.g., human immunoglobulins. The framework may be a human consensus framework or a human consensus framework.

[0135] In some embodiments, the amino acid sequence of SEQ ID NO: 77 is at least about 85%, 86%, or ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, 99%, or 100% sequence identity. V H Anti-CD20 single domain antibodies comprising an H domain are provided. is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Any one of the following: 93%, 94%, 95%, 96%, 97%, 98%, or 99% V has the identity of H The H sequence may contain substitutions (e.g., conservative substitutions), insertions, or or a deletion thereof, but containing the sequence thereof, binds to CD20. In some embodiments, a total of 1 to 10 amino acids are selected from the group consisting of SEQ ID NO: 77 amino acid substitutions, insertions, and / or deletions in the sequence. In some embodiments, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., Optionally, the anti-CD20 single domain antibody may include post-translational modifications of its sequence. The amino acid sequence of SEQ ID NO: 77 is included.

[0136] In some embodiments, a V has the amino acid sequence of SEQ ID NO: 77. H Contains the H domain An isolated anti-CD20 single domain antibody is provided. In some embodiments, the antibody comprises SEQ ID NO: A polypeptide comprising the amino acid sequence of SEQ ID NO:77 is provided.

[0137] In some embodiments, functional epitopes are identified by combinatorial alanine scanning In this process, the sequence can be mapped by combinatorial alanine scanning. Using a coding strategy, we identified the CD20 protein required for interaction with anti-CD20 single domain antibodies. In some embodiments, an epitope can identify an amino acid in a protein. Using the crystal structure of an anti-CD20 single domain antibody in the endothelial conformation and bound to CD20, In some embodiments, the present application provides the Specific for the same epitope as any one of the provided anti-CD20 single domain antibodies For example, in some embodiments, an antibody that binds to the amino acid sequence of SEQ ID NO: 77 is provided. Antibodies are provided that bind to the same epitope as an anti-CD20 single domain antibody comprising the sequence.

[0138] In some embodiments, the present application provides a method for the production of an anti-CD20 single domain antibody described herein. an anti-CD20 antibody that specifically binds to CD20 in competition with any one of the above, or In some embodiments, competitive binding is measured by ELISA. For example, in some embodiments, the amino acid sequence of SEQ ID NO: 77 can be determined using an assay. Antibody specifically binding to CD20 in competition with anti-CD20 single domain antibody containing the amino acid sequence The body is provided.

[0139] In some embodiments, any of the anti-CD20 single domain antibodies described above Anti-CD20 antibodies or antigen binding proteins comprising one or more of the following are provided: In certain embodiments, anti-CD20 antibodies may be monoclonal, including camelid, chimeric, humanized, or human antibodies. In some embodiments, the anti-CD20 antibody is an antibody fragment, e.g., a clonal antibody. For example, V H In some embodiments, the anti-CD20 antibody is an IgG H fragment. full-length heavy chains containing the Fc region of any antibody class or isotype, such as IgG1 or IgG4 In some embodiments, the Fc region is an antibody with reduced or minimized effect. It has a target function.

[0140] In some embodiments, the anti-CD20 antibody (anti- CD20 single domain antibodies) or antigen-binding proteins that meet the criteria listed in the "Antibody Characteristics" below. Any of the features described in sections 1 to 7 may be incorporated alone or in combination.

[0141] In some embodiments, the anti-CD20 antibodies described above (anti-CD20 single domain antibodies) Isolated nucleic acids encoding any one of the following (e.g., nucleotide sequences): In one embodiment, an isolated nucleic acid encoding an anti-CD20 single domain antibody is provided, and the nucleic acid The acid has at least about 85%, 86%, 87%, 88%, 89%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 102. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% sequence identity. In embodiments, an isolated nucleic acid is provided comprising the nucleic acid sequence of SEQ ID NO: 102. In some embodiments, vectors (e.g., expression vectors) containing such nucleic acids are provided. In some embodiments, a host cell containing such nucleic acid is provided. A method for producing an anti-CD20 antibody is provided, the method comprising: Host cells containing nucleic acid encoding the antibody are cultured under conditions suitable for expression of the anti-CD20 antibody. and optionally recovering the anti-CD20 antibody from the host cells (or host cell culture medium). Includes toto.

[0142] Anti-BCMA single domain antibodies In one aspect, the present application provides an isolated single antibody that specifically binds to BCMA, such as human BCMA. In some embodiments, the anti-BCMA single domain antibody is a B In some embodiments, the anti-BCMA single domain antibody modulates BCMA activity. It is an antagonist antibody.

[0143] B-cell maturation antigen (BCMA), also known as CD269, binds to the tumor necrosis factor receptor superfamily. family member, i.e., TNFRSF17 (Thompson et al. l., J. Exp. Medicine, 192(1):129-135, 2000). Hi BCMA is expressed almost exclusively on plasma cells and multiple myeloma cells (e.g., No. vak et al.,Blood,103(2):689-694,2004;Ner i et al.,Clinical Cancer Research,73(19) :5903-5909, Felix et al.,Mol.Oncology,9(7 BCMA is a B cell activating factor (BAF) FF) and proliferation including l igand) (APRIL) (see, e.g., Mackay et al. l., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005). BCMA is a key regulator of immunity against multiple myeloma. High-affinity antibodies targeting BCMA and its natural ligands may be suitable tumor antigen targets for immunotherapy. Anti-BCMA single domain antibodies can block the binding between BAFF and APRIL. , in combination with cellular immunotherapy using CAR-T cells, e.g., to target tumor cells This can enhance the cytotoxic effect against

[0144] In some embodiments, one, two, or all three of the amino acid sequences of SEQ ID NO: 78 In some embodiments, an anti-BCMA single domain antibody is provided comprising the CDRs of Anti-BCMA single antibody containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 79 In some embodiments, domain antibodies are provided, comprising one of the amino acid sequences of SEQ ID NO: 80. Anti-BCMA single domain antibodies comprising one, two, or all three CDRs are provided. In some embodiments, one, two, or all three Cs of the amino acid sequence of SEQ ID NO: 81 In some embodiments, an anti-BCMA single domain antibody is provided, the anti-BCMA single domain antibody comprising a DR. Anti-BCMA single domain antibodies containing one, two, or all three CDRs of the amino acid sequence of No. 82 In some embodiments, an antibody is provided that has one of the amino acid sequences of SEQ ID NO: 83: Anti-BCMA single domain antibodies comprising two or all three CDRs are provided. In some embodiments, one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 84 In some embodiments, an anti-BCMA single domain antibody is provided comprising SEQ ID NO: 8 Anti-BCMA single domains containing one, two, or all three CDRs of the 5 amino acid sequence In some embodiments, antibodies are provided that contain one, two or more of the amino acid sequence of SEQ ID NO: 86. Anti-BCMA single domain antibodies comprising one or all three CDRs are provided. In embodiments, the amino acid sequence of SEQ ID NO: 87 is a CDR sequence comprising one, two, or all three CDRs. In some embodiments, an anti-BCMA single domain antibody is provided, comprising the sequence of SEQ ID NO: 88. Anti-BCMA single domain antibodies containing one, two, or all three CDRs of the amino acid sequence In some embodiments, the anti-BCMA single domain antibody is a camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. In embodiments, the anti-BCMA single domain antibody comprises an acceptor human framework, e.g. For example, a human immunoglobulin framework or a human consensus framework .

[0145] In some embodiments, (a) the amino acid sequence selected from SEQ ID NOs: 7-17 (b) CDR1, (b) CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 18 to 28, and (c) a small amount selected from CDR3s comprising an amino acid sequence selected from SEQ ID NOs: 29 to 39; Anti-BCMA single domains containing at least one, at least two, or all three CDRs In some embodiments, the anti-BCMA single domain antibody is a Camelidae antibody. In some embodiments, the anti-BCMA single domain antibody is humanized. In some embodiments, the anti-BCMA single domain antibody comprises an acceptor human framework , for example, a human immunoglobulin framework or a human consensus framework include.

[0146] In some embodiments, (a) an amino acid sequence selected from SEQ ID NOs: 7-17 and at least one At most about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (b) a CDR1 having one sequence identity with an amino acid sequence selected from SEQ ID NOs: 18 to 28; Column and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (c) a CDR2 having sequence identity with any one of SEQ ID NOs: 29 to 39; 85%, 86%, 87%, 88%, 89%, 90%, 91%, or more of the amino acid sequence %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % sequence identity of any one of the CDR3s of the anti-B CMA single domain antibodies are provided. In some embodiments, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, CDRs with any one of 96%, 97%, 98%, or 99% identity , which contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but Anti-BCMA single domain antibodies containing the BCMA sequence retain the ability to bind to BCMA. In some embodiments, the anti-BCMA single domain antibody is an affinity matured antibody. In some embodiments, the anti-BCMA single domain antibody is camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. Single domain antibodies are constructed using acceptor human frameworks, e.g., human immunoglobulin frameworks. framework or human consensus framework.

[0147] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 29. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. BCMA single domain antibodies can be constructed using acceptor human frameworks, e.g., human immunoglobulin G Includes the Brin framework or the human consensus framework.

[0148] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8; (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 30. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. BCMA single domain antibodies can be constructed using acceptor human frameworks, e.g., human immunoglobulin G Includes the Brin framework or the human consensus framework.

[0149] In some embodiments, the CDR1 comprises: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 31. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. BCMA single domain antibodies can be constructed using acceptor human frameworks, e.g., human immunoglobulin G Includes the Brin framework or the human consensus framework.

[0150] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 10; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 32. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0151] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0152] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 34. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0153] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 35. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0154] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14; (b) (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 25, (d) CDR3 comprising the amino acid sequence of SEQ ID NO: 36 Anti-BCMA single domain antibodies comprising three CDRs, including CDR3, are provided. In some embodiments, the anti-BCMA single domain antibody is camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. CMA single domain antibodies are constructed using acceptor human frameworks, e.g., human immunoglobulins. Contains the Lin framework or the human consensus framework.

[0155] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 37 Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0156] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 16; (b) (c) CDR2 comprising the amino acid sequence of SEQ ID NO: 27, (d) CDR3 comprising the amino acid sequence of SEQ ID NO: 38 Anti-BCMA single domain antibodies comprising three CDRs, including CDR3, are provided. In some embodiments, the anti-BCMA single domain antibody is camelid. In some embodiments, the anti-BCMA single domain antibody is humanized. CMA single domain antibodies are constructed using acceptor human frameworks, e.g., human immunoglobulins. Contains the Lin framework or the human consensus framework.

[0157] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. Anti-BCMA single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-BCMA single domain antibody is a camelid. In embodiments, the anti-BCMA single domain antibody is humanized. Anti-BCMA single domain antibodies may be constructed using acceptor human frameworks, e.g., human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0158] In some embodiments, the amino acid sequence is selected from SEQ ID NOs: 78-88. Also about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% V with sequence identity H Anti-BCMA single domain antibodies comprising the H domain are provided. In some embodiments, at least about 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% V has one of the identities H The H sequence is a sequence of substitutions (e.g., anti-BCMA single domain antibodies containing the sequence, but including conservative substitutions), insertions, or deletions In some embodiments, a total of 1 to 10 antibodies retain the ability to bind to BCMA. The amino acid sequence inserted is replaced with an amino acid sequence selected from SEQ ID NOs: 78 to 88. In some embodiments, substitutions, insertions, or deletions occurs in a region outside the CDR (i.e., within the FR). Optionally, an anti-BCMA single domain The antibody has an amino acid sequence selected from SEQ ID NOs: 78 to 88, including post-translational modifications of that sequence. Contains columns.

[0159] In some embodiments, a V has the amino acid sequence of SEQ ID NO: 78. H Contains the H domain An isolated anti-BCMA single domain antibody is provided. In some embodiments, the antibody has SEQ ID NO: In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 78 is provided. V having amino acid sequence number 79 H Isolated anti-BCMA single domain antibody containing the H domain In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 79 is provided. In some embodiments, a peptide is provided having the amino acid sequence of SEQ ID NO: 80. V H Isolated anti-BCMA single domain antibodies comprising an H domain are provided. In embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO: 80. In one embodiment, V has the amino acid sequence of SEQ ID NO: 81. H Isolated H domain-containing Anti-BCMA single domain antibodies are provided. In some embodiments, the antibody of SEQ ID NO: 81 In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 82 is provided. V with amino acid sequence H An isolated anti-BCMA single domain antibody containing the H domain is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 82 is provided. In some embodiments, a V having the amino acid sequence of SEQ ID NO: 83 is provided. H H-dom In some embodiments, an isolated anti-BCMA single domain antibody is provided, comprising: In some embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO: 83. In the example, V has the amino acid sequence of SEQ ID NO: 84. H Isolated anti-BCMA containing H domain In some embodiments, single domain antibodies are provided, the amino acid sequence of which is set forth in SEQ ID NO: 84. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 85 is provided. V with columns H An isolated anti-BCMA single domain antibody comprising an H domain is provided. In some embodiments, a polypeptide is provided that comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the V has the amino acid sequence of SEQ ID NO: 86. H Contains the H domain An isolated anti-BCMA single domain antibody is provided. In some embodiments, the antibody has SEQ ID NO: In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 86 is provided. V having the amino acid sequence number 87 H Isolated anti-BCMA single domain antibody containing the H domain In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 87 is provided. In some embodiments, a peptide is provided having the amino acid sequence of SEQ ID NO: 88. V H Isolated anti-BCMA single domain antibodies comprising an H domain are provided. In embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO:88.

[0160] In some embodiments, functional epitopes are identified by combinatorial alanine scanning In this process, the sequence can be mapped by combinatorial alanine scanning. Using a cloning strategy, we identified the BCMA protein required for interaction with anti-BCMA single domain antibodies. In some embodiments, an epitope can identify an amino acid in a protein. Using the crystal structure of an anti-BCMA single domain antibody in the endothelial conformation and bound to BCMA, In some embodiments, the present application provides the specifically binds to the same epitope as any of the anti-BCMA single domain antibodies provided. For example, in some embodiments, an antibody comprising the amino acid sequence of SEQ ID NO: 78 is provided. Antibodies that bind to the same epitope as anti-BCMA single domain antibodies comprising In some embodiments, an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 79; Antibodies that bind to the same epitope are provided. In some embodiments, the antibody of SEQ ID NO: 80 Antibodies that bind to the same epitope as anti-BCMA single domain antibodies containing the same amino acid sequence are provided. In some embodiments, an anti-BCMA single antibody comprises the amino acid sequence of SEQ ID NO: 81. Antibodies that bind to the same epitope as the main antibody are provided. Binds to the same epitope as the anti-BCMA single domain antibody containing the amino acid sequence of sequence number 82. In some embodiments, an anti-B antibody is provided that comprises the amino acid sequence of SEQ ID NO: 83. Antibodies that bind to the same epitope as the CMA single domain antibody are provided. In one embodiment, the antibody has the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, antibodies that bind to the polypeptide of SEQ ID NO: 85 are provided. Antibodies that bind to the same epitope as anti-BCMA single domain antibodies containing the sequences are provided. In some embodiments, the anti-BCMA single domain antibody comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, an antibody is provided that binds to the same epitope as SEQ ID NO: 87. The present invention provides an antibody that binds to the same epitope as an anti-BCMA single domain antibody containing the amino acid sequence In some embodiments, an anti-BCMA antibody comprising the amino acid sequence of SEQ ID NO: 88 is provided. Antibodies that bind to the same epitope as the domain antibodies are provided.

[0161] In some embodiments, the present application provides methods for the production of anti-BCMA single domain antibodies described herein. an anti-BCMA antibody that specifically binds to BCMA in competition with any one of the above, or In some embodiments, competitive binding is measured by ELISA. For example, in some embodiments, the amino acid sequence of SEQ ID NO: 78 can be determined using an assay. Antibodies that specifically bind to BCMA in competition with anti-BCMA single domain antibodies containing the amino acid sequence In some embodiments, an anti-BCM antibody comprises the amino acid sequence of SEQ ID NO: 79. Antibodies that specifically bind to BCMA in competition with a single domain antibody are provided. In one embodiment, the antibody competes with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, an antibody that specifically binds to BCMA is provided, the antibody comprising SEQ ID NO: It specifically binds to BCMA in competition with an anti-BCMA single domain antibody containing an 81 amino acid sequence. In some embodiments, an antibody comprising the amino acid sequence of SEQ ID NO: 82 is provided. Antibodies are provided that specifically bind to BCMA in competition with anti-BCMA single domain antibodies. In some embodiments, an anti-BCMA single domain antibody comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, antibodies that specifically bind to BCMA in competition with the human antibody are provided. A compound that specifically binds to BCMA and competes with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 84. Antibodies that bind to the amino acid sequence of SEQ ID NO: 85 are provided. The present invention provides an antibody that specifically binds to BCMA by competing with an anti-BCMA single domain antibody containing a nucleotide sequence. In some embodiments, an anti-BCMA single antibody comprises the amino acid sequence of SEQ ID NO: 86. Antibodies that specifically bind to BCMA in competition with the main antibody are provided. In this embodiment, the BCMA antibody is capable of competing with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 87. Antibodies that specifically bind to MA are provided. In some embodiments, the antibody of SEQ ID NO: 88 Antibodies that specifically bind to BCMA in competition with anti-BCMA single domain antibodies containing the amino acid sequence The body is provided.

[0162] In some embodiments, any of the anti-BCMA single domain antibodies described above Anti-BCMA antibodies or antigen binding proteins comprising one or more of the following are provided: In certain embodiments, anti-BCMA antibodies may be monoclonal, including camelid, chimeric, humanized, or human antibodies. In some embodiments, the anti-BCMA antibody is an antibody fragment, e.g., a clonal antibody. For example, V H In some embodiments, the anti-BCMA antibody is an IgG full-length heavy chains containing the Fc region of any antibody class or isotype, such as IgG1 or IgG4 In some embodiments, the Fc region is an antibody with reduced or minimized effect. It has a target function.

[0163] In some embodiments, the anti-BCMA antibody (anti- BCMA single domain antibodies) or antigen-binding proteins meet the first of the "Antibody Characteristics" below. Any of the features described in sections 1 to 7 may be incorporated alone or in combination.

[0164] In some embodiments, the anti-BCMA antibodies described above (anti-BCMA single domain antibodies) Isolated nucleic acids encoding any one of the following (e.g., nucleotide sequences): In one embodiment, an isolated nucleic acid encoding an anti-BCMA single domain antibody is provided, the nucleic acid being The nucleic acid has at least about 85% affinity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 103-113. ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95% , 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the nucleic acid sequence comprises a sequence having the group consisting of SEQ ID NOs: 103-113. An isolated nucleic acid comprising a selected nucleic acid sequence is provided. In some embodiments, a vector (e.g., an expression vector) is provided that comprises the nucleic acid. In some embodiments, the anti-BCMA antibody is Methods of making are provided, which methods comprise using nucleic acid encoding the anti-BCMA antibodies provided above. Culturing a host cell comprising the compound under conditions suitable for expression of an anti-BCMA antibody, and optionally, and recovering the CMA antibody from the host cell (or host cell culture medium).

[0165] Anti-CD38 single domain antibody In one aspect, the present application provides an isolated single antibody that specifically binds to CD38, such as human CD38. In some embodiments, the anti-CD38 single domain antibody is In some embodiments, the anti-CD38 single domain antibody modulates CD38 activity. It is an antagonist antibody.

[0166] CD38 associates with cell surface receptors and mediates cytoplasmic Ca 2+ Controls the flow of lymphocytes and Konopl is a type II transmembrane glycoprotein that mediates signal transduction in the spinal cord. eva et al., J Immunol, 161:4702-8, 1998, Dea glio et al., Blood, 109:5390-8, 2007). Human CD3 8 is highly and uniformly expressed in myeloma cells and in normal lymphoid and myeloid cells as well as non-hematopoietic cells. It is expressed at relatively low levels in some tissues of origin, making it a potential candidate for the treatment of myeloma. Targeting (e.g., Lin et al., Am J Clin Pathol, 20 04,121:482, HMLokhorst et al., New Eng.J (See .Med.,2015,373:13).

[0167] In some embodiments, one, two, or all three of the amino acid sequences of SEQ ID NO: 89 In some embodiments, an anti-CD38 single domain antibody is provided, comprising the CDRs of Anti-CD38 monoclonal antibodies containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 90 In some embodiments, domain antibodies are provided, comprising one of the amino acid sequences of SEQ ID NO: 91. Anti-CD38 single domain antibodies comprising one, two, or all three CDRs are provided. In some embodiments, one, two, or all three Cs of the amino acid sequence of SEQ ID NO: 92 are In some embodiments, an anti-CD38 single domain antibody is provided, comprising SEQ ID NO: Anti-CD38 single domain antibodies containing one, two, or all three CDRs of the amino acid sequence of No. 93 In some embodiments, an antibody is provided that has one of the amino acid sequences of SEQ ID NO: 94: Anti-CD38 single domain antibodies comprising two or all three CDRs are provided. In some embodiments, one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 95 In some embodiments, an anti-CD38 single domain antibody is provided, comprising SEQ ID NO: 9 Anti-CD38 single domains containing one, two, or all three CDRs of the six amino acid sequences In some embodiments, antibodies are provided that contain one, two or more of the amino acid sequence of SEQ ID NO: 97. Anti-CD38 single domain antibodies comprising one or all three CDRs are provided. In embodiments, the amino acid sequence of SEQ ID NO: 98 is a CDR sequence comprising one, two, or all three CDRs. In some embodiments, an anti-CD38 single domain antibody is provided, comprising the sequence of SEQ ID NO: 99. Anti-CD38 single domain antibodies containing one, two, or all three CDRs of the amino acid sequence In some embodiments, one, two, or all of the amino acid sequences of SEQ ID NO: 100 are provided. Alternatively, anti-CD38 single domain antibodies comprising all three CDRs are provided. In some embodiments, the anti-CD38 single domain antibody is camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. Single domain antibodies are constructed using acceptor human frameworks, e.g., human immunoglobulin frameworks. framework or human consensus framework.

[0168] In some embodiments, (a) a nucleic acid sequence comprising an amino acid sequence selected from SEQ ID NOs: 40-51. (b) a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 52 to 63; and and (c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 64 to 75. Anti-CD38 single domain antibodies containing at least one, at least two, or all three CDRs In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. In some embodiments, the anti-CD38 single domain antibody is an antibody derived from an acceptor human framework. framework, e.g., a human immunoglobulin framework or a human consensus framework Includes:

[0169] In some embodiments, (a) an amino acid sequence selected from SEQ ID NOs: 40-51 and at least one At least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% (b) a CDR1 having one sequence identity with an amino acid selected from SEQ ID NOs: 52 to 63; Sequence and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of (c) a CDR2 having sequence identity with any one of SEQ ID NOs: 64 to 75; and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 0% sequence identity to any one of the CDRs. CD38 single domain antibodies are provided. In some embodiments, at least about 85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95% , 96%, 97%, 98%, or 99% identity to any one of the CDRs contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but Anti-CD38 single domain antibodies containing the sequence retain the ability to bind to CD38. In some embodiments, the anti-CD38 single domain antibody is camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The D38 single domain antibody is constructed using an acceptor human framework, e.g., a human immunoglobulin. In some embodiments, the ribonucleotides may be a ribonucleotide or a nucleotide sequence. In the present case, the anti-CD38 single domain antibody is an affinity matured antibody.

[0170] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 64. Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0171] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 41; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 65. Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0172] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 42; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 66 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0173] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 43; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 67 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0174] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 68 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0175] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 45; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 69 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0176] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 70 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0177] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 47; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 71. Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0178] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 48; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 72 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0179] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 49; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 73 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0180] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 50; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 74 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0181] In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 51; (b) (c) a CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and (d) a CDR3 comprising the amino acid sequence of SEQ ID NO: 75 Anti-CD38 single domain antibodies comprising three CDRs are provided, including a CDR3 comprising: In some embodiments, the anti-CD38 single domain antibody is a camelid. In some embodiments, the anti-CD38 single domain antibody is humanized. The anti-CD38 single domain antibody may be constructed using an acceptor human framework, e.g., a human immunoglobulin G It includes a globulin framework or a human consensus framework.

[0182] In some embodiments, the amino acid sequence is at least one selected from SEQ ID NOs: 89-100. Approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 One of the following: 4%, 95%, 96%, 97%, 98%, 99%, or 100% V with sequence identity of H Anti-CD38 single domain antibodies comprising the H domain are provided. In some embodiments, at least about 85%, 86%, 87%, 88%, 89%, 90%, %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% V having any one of the identities H The H sequence is compared to the reference sequence to identify substitutions (e.g. , conservative substitutions), insertions, or deletions, but containing the sequence of an anti-CD38 single domain antibody. In some embodiments, a total of 1 to 10 The amino acid sequence is selected from SEQ ID NOs: 89 to 100, and the insertion In some embodiments, substitutions, insertions, or deletions are present. The deletion occurs in a region outside the CDR (i.e., within the FR). The domain antibody is an amino acid sequence selected from SEQ ID NOs: 89 to 100, including post-translational modifications of that sequence. Contains the amino acid sequence.

[0183] In some embodiments, a V has the amino acid sequence of SEQ ID NO: 89. H Contains the H domain An isolated anti-CD38 single domain antibody is provided. In some embodiments, the antibody comprises SEQ ID NO: In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 89 is provided. V having amino acid sequence number 90 H Isolated anti-CD38 single domain containing the H domain In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 90 is provided. In some embodiments, a peptide is provided having the amino acid sequence of SEQ ID NO: 91. V H Isolated anti-CD38 single domain antibodies comprising the H domain are provided. In embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO: 91. In one embodiment, V has the amino acid sequence of SEQ ID NO: 92. H Isolated H domain-containing Anti-CD38 single domain antibodies are provided. In some embodiments, the antibody of SEQ ID NO: 92 In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 93 is provided. V with amino acid sequence H An isolated anti-CD38 single domain antibody containing the H domain is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 93 is provided. In some embodiments, a V having the amino acid sequence of SEQ ID NO: 94 is provided. H H-dom In some embodiments, an isolated anti-CD38 single domain antibody is provided, comprising: In some embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO: 94. V having the amino acid sequence of SEQ ID NO: 95 H Isolated anti-CD38 containing the H domain In some embodiments, single domain antibodies are provided, the amino acid sequence of which is set forth in SEQ ID NO: 95. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 96 is provided. V with columns H An isolated anti-CD38 single domain antibody comprising an H domain is provided. In some embodiments, a polypeptide is provided that comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the V has the amino acid sequence of SEQ ID NO: 97. H Contains the H domain An isolated anti-CD38 single domain antibody is provided. In some embodiments, the antibody comprises SEQ ID NO: In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO:97 is provided. V having amino acid sequence number 98 H Isolated anti-CD38 single domain containing the H domain In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 98 is provided. In some embodiments, a peptide is provided having the amino acid sequence of SEQ ID NO: 99. V H Isolated anti-CD38 single domain antibodies comprising the H domain are provided. In embodiments, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO: 99. In one embodiment, V has the amino acid sequence of SEQ ID NO: 100. H Isolated nucleotides containing the H domain In some embodiments, an anti-CD38 single domain antibody is provided, comprising SEQ ID NO: 100. A polypeptide comprising the amino acid sequence of

[0184] In some embodiments, functional epitopes are identified by combinatorial alanine scanning In this process, the sequence can be mapped by combinatorial alanine scanning. Using a cloning strategy, we identified the CD38 protein required for interaction with anti-CD38 single domain antibodies. In some embodiments, an epitope can identify an amino acid in a protein. Using the crystal structure of an anti-CD38 single domain antibody in the endothelial conformation and bound to CD38, In some embodiments, the present application provides the specifically binds to the same epitope as any of the provided anti-CD38 single domain antibodies. For example, in some embodiments, an antibody comprising the amino acid sequence of SEQ ID NO: 89 is provided. Antibodies that bind to the same epitope as anti-CD38 single domain antibodies comprising: In some embodiments, an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 90; Antibodies that bind to the same epitope are provided. In some embodiments, the antibody of SEQ ID NO: 91 Antibodies that bind to the same epitope as anti-CD38 single domain antibodies containing the same amino acid sequence are provided. In some embodiments, an anti-CD38 single antibody comprises the amino acid sequence of SEQ ID NO: 92. Antibodies that bind to the same epitope as the main antibody are provided. Binds to the same epitope as the anti-CD38 single domain antibody containing the amino acid sequence of SEQ ID NO: 93 In some embodiments, an anti-C antibody is provided that comprises the amino acid sequence of SEQ ID NO: 94. Antibodies that bind to the same epitope as the D38 single domain antibody are provided. In this form, the antibody has the same epitope as an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, antibodies that bind to the polypeptide of SEQ ID NO: 96 are provided. Antibodies that bind to the same epitope as anti-CD38 single domain antibodies containing the sequences are provided. In some embodiments, an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an antibody is provided that binds to the same epitope as SEQ ID NO: 98. The present invention provides an antibody that binds to the same epitope as an anti-CD38 single domain antibody containing the amino acid sequence of In some embodiments, an anti-CD38 monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 99 is provided. Antibodies that bind to the same epitope as a domain antibody are provided. In some embodiments, Binds to the same epitope as the anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 100 Antibodies that bind to the antibody are provided.

[0185] In some embodiments, the present application provides methods for the production of anti-CD38 single domain antibodies described herein. an anti-CD38 antibody that specifically binds to CD38 in competition with any one of the above, or In some embodiments, competitive binding is measured by ELISA. For example, in some embodiments, the amino acid sequence of SEQ ID NO: 89 can be determined using an assay. Antibody specifically binding to CD38 in competition with anti-CD38 single domain antibody containing the amino acid sequence In some embodiments, an anti-CD3 antibody comprising the amino acid sequence of SEQ ID NO: 90 is provided. 8. Antibodies that specifically bind to CD38 in competition with single domain antibodies are provided. In one embodiment, the antibody competes with an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, an antibody that specifically binds to CD38 is provided, the antibody being represented by SEQ ID NO: It specifically binds to CD38 in competition with an anti-CD38 single domain antibody containing a 92 amino acid sequence. In some embodiments, an antibody comprising the amino acid sequence of SEQ ID NO: 93 is provided. Antibodies that specifically bind to CD38 in competition with anti-CD38 single domain antibodies are provided. In some embodiments, an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, antibodies that specifically bind to CD38 in competition with the human antibody are provided. A compound that specifically binds to CD38 and competes with an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 95. Antibodies that bind to the amino acid sequence of SEQ ID NO: 96 are provided. Antibodies that specifically bind to CD38 in competition with anti-CD38 single domain antibodies containing the nucleotide sequence are provided. In some embodiments, an anti-CD38 single antibody comprises the amino acid sequence of SEQ ID NO: 97. Antibodies that specifically bind to CD38 in competition with the main antibody are provided. In this embodiment, the CD38 single domain antibody is capable of competing with an anti-CD38 single domain antibody comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, an antibody that specifically binds to SEQ ID NO: 38 is provided. Antibody specifically binding to CD38 in competition with anti-CD38 single domain antibody containing the amino acid sequence In some embodiments, an anti-CD3 antibody is provided that comprises the amino acid sequence of SEQ ID NO: 100. Antibodies are provided that specifically bind to CD38 in competition with the CD38 single domain antibody.

[0186] In some embodiments, any of the anti-CD38 single domain antibodies described above Anti-CD38 antibodies or antigen binding proteins comprising one or more of the following are provided: In certain embodiments, anti-CD38 antibodies may be monoclonal, including camelid, chimeric, humanized, or human antibodies. In some embodiments, the anti-CD38 antibody is an antibody fragment, e.g., a clonal antibody. For example, V H In some embodiments, the anti-CD38 antibody is an IgG H fragment. full-length heavy chains containing the Fc region of any antibody class or isotype, such as IgG1 or IgG4 In some embodiments, the Fc region is an antibody with reduced or minimized effect. It has a target function.

[0187] In some embodiments, the anti-CD38 antibody (anti- CD38 single domain antibodies) or antigen-binding proteins that meet the criteria listed in the "Antibody Characteristics" below. Any of the features described in sections 1 to 7 may be incorporated alone or in combination.

[0188] In some embodiments, the anti-CD38 antibodies described above (anti-CD38 single domain antibodies) Isolated nucleic acids encoding any one of the following (e.g., nucleotide sequences): In one embodiment, an isolated nucleic acid encoding an anti-CD38 single domain antibody is provided, the nucleic acid comprising , at least about 85%, 8 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 114-125. In some embodiments, isolated nucleic acids are provided that contain a nucleic acid sequence that is In some embodiments, vectors (e.g., expression vectors) containing the acid are provided. Host cells containing such nucleic acids are provided. In some embodiments, the anti-CD38 antibody is produced by The present invention provides a method for producing a CD38 antibody comprising the steps of: Culturing host cells containing the anti-CD38 antibody under conditions suitable for expression of the anti-CD3 antibody, and optionally, and recovering the antibody from the host cell (or host cell culture medium).

[0189] Antibody Characteristics 1. Antibody affinity In some embodiments, the antibodies provided herein have a concentration of 1 μM or less, 100 nM or less. , 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001n M or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10- 9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0190] In some embodiments, the Kd is determined by the following assay: Fab or V version of the antibody H Radioactivity using H fragment and its antigen Binding of antibodies to Fab fragments is measured by radiolabeled antigen binding assay (RIA). The solution binding affinity of Fab is determined by binding the Fab to the lowest concentration ( 125 I) Equilibrated with labeled antigen and then bound to an anti-Fab antibody coated plate It is measured by capturing antibodies (see, e.g., Chen et al., J. Mol. (See Biol. 293:865-881 (1999)). To establish the conditions, MICROTITER® multiwell plates (The rmo Scientific) at 5 μg / mL in 50 mM sodium carbonate (pH 9.6) mL of capture anti-Fab antibody (Cappel Labs) overnight, followed by P Block with 2% w / v bovine serum albumin in BS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc No. 269620), 100 pM or 26 pM of[ 125 I] antigen is mixed with serial dilutions of the Fab of interest (Presta et al. al., Cancer Res. 57:4593-4599 (1997) (This is consistent with the evaluation of the EGF antibody, Fab-12). Then, the target Fab was incubated overnight. Incubate for 1 minute, but to ensure equilibrium is achieved, extend this incubation. It may be continued for a longer period of time (e.g., about 65 hours). Transfer to a plate and incubate at room temperature (e.g., 1 hour). Then, remove the solution. and the plates were washed with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, wash with 150 μL / well of scintillant (sc intillant (MICROSCINT-20™, Packard) was added. and the plate was counted on a TOPCOUNT™ gamma counter (Packard) for 10 minutes. The concentration of each Fab that yielded 20% or less of the maximum binding was used in the competitive binding assay. Select for use in

[0191] In some embodiments, the Kd is about 10 response units (RU) on an immobilized antigen CM5 chip. BIACORE®-2000 or BIACORE®-3 000 (BIAcore, Inc., Piscataway, NJ) at 25°C. It is measured using a surface plasmon resonance assay. A trans-biosensor chip (CM5, BIACORE, Inc.) was prepared according to the supplier's instructions. N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride was prepared according to the instructions. Activated with salt (EDC) and N-hydroxysuccinimide (NHS). Dilute the antigen to 5 μg / mL (approximately 0.2 μM) with sodium chloride (pH 4.8). After dilution, the solution was injected at a flow rate of 5 μL / min, resulting in a coupling of approximately 10 response units (RU). After the antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, the Fab or V of the antibody of interest is H2-fold serial dilutions of H (0.78 nM to 500 nM) in 0.05% polysorbate 20 (TWEEN -20 TM ) Injected at a flow rate of approximately 25 μL / min in PBS with surfactant (PBST). The association rate (k on ) and dissociation rate (k off ) is a simple one-to-one Langmuir reaction Using the combined model (BIACORE® evaluation software version 3.2) , calculated by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is k off / k on For example, Chen et al. See, e.g., J. Mol. Biol. 293:865-881 (1999). The on-rate was 10 by the surface plasmon resonance assay described above. 6 M -1 s -1 A place beyond In this case, the on-speed is measured using a stop-flow equipped spectrophotometer (Aviv Instruments) ts) or 8000 Series SLM-AMINCO™ with Stirred Cuvette Increasing concentrations of antigen measured with a spectrophotometer such as a ThermoSpectronic Fluorescence of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of Increase or decrease in light emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) This can be determined by using a fluorescence quenching technique that measures the amount of

[0192] 2. Antibody fragments In some embodiments, the antibodies provided herein are antibody fragments. The antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragment, V H H, as well as other fragments described below. For a review of certain antibody fragments, see Huds See on et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies,v ol.113,Rosenburg and Moore eds.,(Springe r-Verlag, New York), pp. 269-315 (1994). and WO93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,571,895. See also US Pat. No. 87,458. For a discussion of Fab and F(ab')2 fragments with similar in vivo half-lives, see See U.S. Patent No. 5,869,046.

[0193] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. For example, see EP 404,097 and WO 1993 / 01161. No., Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. US A 90:6444-6448 (1993). Triabodies and tetrabodies For details, see Hudson et al., Nat. Med. 9:129-134 ( 2003) is also mentioned.

[0194] Antibody fragments, as described herein, are proteolytic enzymes derived from intact antibodies. as well as production by recombinant host cells (e.g., E. coli or phage) They can be made by a variety of techniques, including but not limited to:

[0195] 3. Chimeric and humanized antibodies In some embodiments, the antibodies provided herein are chimeric antibodies. Chimeric antibodies of the present invention are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al. t al.,Proc.Natl.Acad.Sci.USA,81:6851-685 5 (1984)). In one example, a chimeric antibody contains a non-human variable region (e.g., In a further example, the antibody may comprise a human constant region (variable region derived from a Camelidae species such as a llama, etc.) and a human constant region. Chimeric antibodies are antibodies whose class or subclass has changed from that of the parent antibody. A chimeric antibody is a "class-switched" antibody that has been modified to contain its antigen-binding fragment. include.

[0196] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody The antibody has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, including HVRs, For example, the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are Humanized antibodies are derived from human antibody sequences. Humanized antibodies optionally also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody will be, e.g., In order to restore or improve antibody specificity or affinity, non-human antibodies (e.g., HVR residues) may be modified. The group is replaced with the corresponding residue from the antibody from which the group is derived.

[0197] Humanized antibodies and methods for their production are described, for example, in Almagro and Frans on, Front.Biosci.13:1619-1633(2008), For example, Riechmann et al., Nature 332:323-329 ( 1988), Queen et al., Proc. Nat'l Acad. Sci. U SA 86:10029-10033(1989), U.S. Patent No. 5,821,337, Nos. 7,527,791, 6,982,321, and 7,087,409 ,Kashmiri et al.,Methods 36:25-34(2005)( (SDR (a-CDR) grafting described), Padlan, Mol. Immunol .28:489-498(1991) (describing "surface reconstruction"), Dall'Ac qua et al., Methods 36:43-60(2005) ("FR Shuttle (described in "Fling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cance r,83:252-260(2000) ("Guided selection" approach to FR shuffling) This is further described in (described above).

[0198] Human framework regions that can be used for humanization are identified using the "best fit" method. The framework region selected (e.g., Sims et al. J. Immunol. 151:2296 (1993), specific subunits of the light or heavy chain variable region Framework regions derived from the consensus sequence of human antibodies from the group (e.g., Car ter et al.Proc.Natl.Acad.Sci.USA,89:4285 (1992), and Presta et al. J. Immunol., 151:262 3 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (e.g., Almagro and Fransson, F ront.Biosci.13:1619-1633(2008)). and framework regions (e.g., Ba ca et al.,J.Biol.Chem.272:10678-10684(19 97) and Rosok et al., J. Biol. Chem. 271:22611- 22618 (1996)).

[0199] In some embodiments, single domain antibodies are characterized by the natural affinity of the domain for the antigen. Modifications to reduce its immunogenicity against heterologous species, while not reducing the For example, the antibody variable domains (V) of a llama antibody are humanized. H The amino acid residues of H) were determined. For example, one or more of the Camelidae amino acids in the framework region may be present in the polypeptide. The resulting polypeptide is humanized without losing its typical characteristics. The human consensus sequence was chosen so as not to significantly affect the antigen-binding capacity of the peptide. The humanization of camelid single domain antibodies involves the use of single domain antibodies. It requires the introduction and mutagenesis of a limited number of amino acids within a polypeptide chain. , the introduction of amino acid changes into the two chains (light and heavy chains) and the assembly of both chains. In contrast to humanization of scFv, Fab', (Fab')2, and IgG, which require conservation is.

[0200] V H Single domain antibodies containing H domains can be humanized to have human-like sequences In some embodiments, V as used herein H The FR region of the H domain is H at least about 50%, 60%, 70%, 80%, 90% relative to the framework region; 95% or more amino acid sequence homology. H One exemplary class of H domain is the V H H is Glycid according to Kabat numbering Alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine threonine, tryptophan, methionine, serine, threonine, asparagine, or glutamine an amino acid from the group consisting of tryptophan at position 103 (e.g., L45, etc.); Therefore, polypeptides belonging to this class are characterized by having a position corresponding to the human V H The polypeptide exhibits high amino acid sequence homology to the framework regions, without anticipating any unwanted immune responses from it and without burdening further humanization. It can be administered directly to humans without any need for administration.

[0201] Another exemplary class of humanized camelid single domain antibodies is described in WO03 / 035694. are described and are typically found in conventional antibodies of human origin or from other species, but V from chain antibodies HThe conserved tryptophan residue in The hydrophobic FR2 residues compensate for this loss of hydrophilicity by substitution with the residues Therefore, peptides belonging to these two classes are H For framework areas and the peptides show high amino acid sequence homology with each other, and the peptides prevent unwanted immune responses therefrom. Can be administered directly to humans without any anticipation and without the burden of further humanization .

[0202] 4. Human antibodies In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. n Dijk and van de Winkel,Curr.Opin.Pharm acol. 5:368-74 (2001) and Lonberg, Curr. Opin. I Mmunol. 20:450-459 (2008). Transgenic mice or rats capable of producing antibodies are known in the art. For example, US20090307787A1, US Pat. No. 7, US20150289489A1, US20100122358A1, and WO2 Please refer to 004049794.

[0203] Human antibodies are those that produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. prepared by administering an immunogen to a transgenic animal that has been modified to produce Such animals typically have genes that replace endogenous immunoglobulin loci. They may be present extrachromosomally or randomly integrated into the animal's chromosomes. Such transgenic vectors contain all or part of the human immunoglobulin locus. In mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg See, e.g., Nat. Biotech. 23:1117-1125 (2005). See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (XENOMOU No. 5,770,429 (which describes HUMAB® technology) No. 7,041,870 (KM MOUSE® technology) ), and U.S. Patent Application Publication No. US2007 / 0061900 (which describes VELOCI See also MOUSE® technology (which describes the use of such technology). Human variable regions from a target antibody can be combined with different human constant regions, e.g. It may be further modified.

[0204] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies are described. (See, for example, Kozbor J. Immunol., 133:3001(1 984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications,p p.51-63 (Marcel Dekker, Inc., New York, 1987 ), and Boerner et al., J. Immunol., 147:86(199 1) Regarding human antibodies produced by human B cell hybridoma technology, However, Li et al.,Proc.Natl.Acad.Sci.USA,103: 3557-3562 (2006). Further methods include, for example, Patent No. 7,189,826 (Monoclonal human IgM from hybridoma cell line) (Describes the production of antibodies) and Ni, Xiandai Mianyixue, 26(4) :265-268 (2006) (describing human-human hybridomas) For human hybridoma technology (trioma technology), see Vollm ers and Brandlein,Histology and Histopat hology, 20(3):927-937(2005) and Vollmers and Brandlein,Methods and Findings in Expert imental and Clinical Pharmacology,27(3): 185:91 (2005).

[0205] Human antibodies are derived from Fv clones selected from a human-derived phage display library. They can also be generated by isolating variable domain sequences. The main sequence can be combined with the desired human constant domains. Techniques for selecting antibodies are described below.

[0206] V directed against a specific antigen or target H One technique for obtaining H sequences is and suitably immunizing a transgenic mammal capable of expressing the antibody. That is, the immune response and / or heavy chain antibodies directed against said antigen or target. (as if dripping), said V H The transgene containing (a nucleic acid sequence encoding) the H sequence Obtain a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from the nicked mammal. and then, starting from said sample, carry out the purification step using any suitable technique known per se (as described herein). or hybridoma technology) to identify the antigen or target V oriented towards H For example, for this purpose, WO02 / 085945, WO04 / 049794 and WO06 / 008548, and Ja nssens et al.,Proc.Natl.Acad.Sci.USA.200 6 Oct.10;103(41):15130-5 and heavy chain antibody expressing mice For example, such heavy chain antibody-expressing mice can be generated from natural sources. (Single) variable domains derived from (e.g., human (single) variable domains, camelid (single) variable domains) variable domains or shark (single) variable domains), as well as synthetic or semi-synthetic (single It is possible to express heavy chain antibodies with any suitable (single) variable domain, such as a This can be done.

[0207] 5. Library-derived Antibodies The antibodies of the present application may be subjected to combinatorial screening for antibodies having the desired activity(ies). For example, the vectors can be isolated by screening phage display libraries. generating a library and screening such library for antibodies possessing desired binding characteristics. Various methods for screening are known in the art. ogenboom et al.in Methods in Molecular B iology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and McCaffer, for example. ty et al., Nature 348:552-554, Clackson et al. al.,Nature 352:624-628(1991);Marks et a l., J. Mol. Biol. 222:581-597 (1992), Marks an d Bradbury,Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ , 2003), Sidhu et al., J.Mol.Biol.338(2):29 9-310(2004);Lee et al.,J.Mol.Biol.340(5) :1073-1093(2004);Fellouse,Proc.Natl.Acad .Sci.USA 101(34):12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2):119-1 32 (2004). To construct a single domain antibody library, This method is described, for example, in US Pat. No. 7,371,849.

[0208] In certain phage display methods, V H and V L The gene repertoire is polymerized They are cloned separately by PCR and then isolated in a phage library. This is then randomly recombined, as described in Winter et al., Ann. Rev. As described in Immunol., 12:433-455 (1994), antigen-binding fragments Phages can be screened for, typically, single chain Fv (scF v) Disclose antibody fragments either as fragments or Fab fragments. Libraries derived from immunized sources can be generated without the need for hybridoma construction. , providing high affinity antibodies to the immunogen. Alternatively, Griffiths et al., E As described in MBO J.12:725-734(1993), naive repart Li has been cloned (e.g., from a human) to produce a broad range of non-self antibodies without immunization. It is possible to provide a single source of antibodies against both the original and self-antigens. oom and Winter, J.Mol.Biol.,227:381-388(1 Stem cell-derived unrearranged V gene fragments were used as described in (992). Cloning and characterization of highly variable Cs using PCR primers containing random sequences The DR3 region is encoded by naive librarian by achieving rearrangement in vitro. The libraries can also be produced synthetically. Patents Describing Human Antibody Phage Libraries Publications include, for example, U.S. Pat. No. 5,750,373 and U.S. Pat. No. 05 / 0079574, No. 2005 / 0119455, No. 2005 / 02660 No. 00, No. 2007 / 0117126, No. 2007 / 0160598, No. 20 Nos. 07 / 0237764, 2007 / 0292936, and 2009 / 000 No. 2360 is one example.

[0209] Antibodies or antibody fragments isolated from a human antibody library are referred to herein as It is considered a human antibody or human antibody fragment.

[0210] 6. Multispecific antibodies In some embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In some embodiments, one binding specificity is an antibody that binds to CD19, CD 20, BCMA, and CD38, and the other is against an antigen selected from the group consisting of In some embodiments, the bispecific antibody is directed against CD19 two different epitopes of an antigen selected from the group consisting of CD20, BCMA, and CD38. Bispecific antibodies can be used to bind to CD19, CD20, and BC a cytotoxic agent against cells expressing an antigen selected from the group consisting of MA and CD38; can also be localized.

[0211] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. One technique for producing bispecific antibodies is to combine two immunoglobulins with different specificities. Recombinant co-expression of a guanine heavy chain-light chain pair (Milstein and Cuello, Nature e 305:537(1983)), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "Knob-in-hole" operation (see, e.g., U.S. Pat. No. 5,731,168) is cited. Multispecific antibodies can be used to manipulate electrostatic steering effects, but are not limited to these. (WO2009 / 089004A1), and Crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,988). No. 0, and Brennan et al., Science, 229:81 (1985) ), using leucine zippers to produce bispecific antibodies (e.g. , Kostelny et al., J. Immunol., 148(5):1547- 1553 (1992)), bispecific antibodies using "diabody" technology. Producing somatic fragments (e.g., Hollinger et al., Proc .Natl.Acad.Sci.USA, 90:6444-6448(1993)), and and single-chain Fv (sFv) dimers (e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and Mie Preparing specific antibodies (e.g., Tutt et al. J. Immunol. 14 7:60 (1991), as well as methods for producing polypeptides including tandem single domain antibodies. (See, e.g., U.S. Patent Application No. 20110028695 and Conrath et al. See al. J. Biol. Chem., 2001;276(10):7346-50 Three or more functional antigens, including "Octopus antibodies," can also be produced by Engineered antibodies having binding sites are also included herein (see, e.g., US2006 / 002 (See 5576A1).

[0212] 7. Antibody variants In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be made by introducing appropriate modifications into the nucleic acid sequence encoding the antibody. Such modifications can be prepared by introducing nucleotides into the nucleotide sequence or by peptide synthesis. For example, deletions from, and / or insertions into, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution to arrive at the final construct may be used. can be performed, provided that the final construct possesses the desired properties, e.g., antigen binding. The matter is as follows.

[0213] a) Substitution, insertion, and deletion variants In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are , shown under the heading "Preferred Substitutions" in Table 3. More substantial changes are shown under the heading "Preferred Substitutions" in Table 3. and further below with reference to amino acid side chain classes, as provided under the heading "Exemplary Substitutions." Amino acid substitutions can be introduced into the antibody of interest to improve the product's desired activity, e.g. , retain / improve antigen binding, reduce immunogenicity, or improve ADCC or CDC can be screened. [Table 3]

[0214] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe.

[0215] Non-conservative substitutions will involve an exchange of one member of one of these classes with another class. This will be accompanied.

[0216] Certain types of substitution variants involve substituting one or more of the hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the variants (s) resulting from selection for further testing have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parental antibody and / or have certain biological properties of the parental antibody that are substantially retained. Exemplary substitution variants are, for example , affinity matured antibodies that can be conveniently generated using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more HVR residues are mutated , the mutant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity ).

[0217] Modifications (e.g., substitutions) may be made within the HVRs, for example, to improve antibody affinity . Such modifications may be made to HVR "hotspots", i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (e.g., Chowdhury, Me thods Mol.Biol.207:179-196(2008)), and / or to the SDR (a-CDR), and the resulting variant V H or V L is tested for binding affinity. Affinity maturation by construction of a secondary library and reselection therefrom For example, Hoogenboom et al. in Methods in Mo lecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturation, various methods (e.g., error-prone PC R, chain shuffling, or oligonucleotide-directed mutagenesis) , diversity is introduced into the variable genes selected for maturation. A secondary library is then This library is then screened to identify any with the desired affinity. Another method for introducing diversity is to identify antibody variants by targeting several HVR residues (e.g. This involves an HVR-directed approach in which multiple residues (4-6 residues at a time) are randomized. HVR residues involved in the In many cases, CDR-H3 and CDR-L3 are particularly Become a target.

[0218] In some embodiments, substitutions, insertions, or deletions are made such that such modifications do not bind to the antigen of the antibody. may occur within one or more HVRs, as long as it does not substantially reduce the ability of the Conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce compatibility Such modifications may be made within HVRs, in HVR "hot spots" or outside the CDRs. The variant V provided above can be H In some embodiments of the H sequence, each HVR is Either unaltered or with no more than one, two, or three amino acid substitutions It's either one or the other.

[0219] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is the "Ala This is called "inscanning mutagenesis" and was published by Cunningham and Wells (1 989) Science, 244: 1081-1085. target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) The residues or groups are identified and neutral or negatively charged amino acids (e.g., alanine or were replaced by polyalanine to determine whether the antibody's interaction with the antigen was affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the first substitution. Alternatively, or in addition, antigen-antibody complexes may be used to identify contact points between the antibody and antigen. The crystal structure of the combined residues. Can such contact residues and neighboring residues be targeted as candidates for substitution? Mutants can be screened to determine whether they have the desired properties. can be determined.

[0220] Amino acid sequence insertions can range from one residue to polypeptides containing 100 or more residues. Amino- and / or carboxyl-terminal fusions with lengths ranging from 100 to 2000, as well as single or intrasequence insertions of multiple amino acid residues. Examples of terminal insertions include N-terminal amino acid Other insertional variants of antibody molecules include antibodies containing thionyl residues. For example, for ADEPT) or N-linking of antibodies to polypeptides that increase the serum half-life of the antibody. Terminal or C-terminal fusions are included.

[0221] b) Glycosylation variants In some embodiments, the antibodies provided herein are glycosylated to the extent that the antibody is glycosylated. The antibody may be modified to increase or decrease its glycosylation level. Deletions are made by altering the amino acid sequence so that one or more glycosylation sites are created or removed. This can be conveniently achieved by

[0222] If the antibody contains an Fc region, the carbohydrate attached to it may be modified. Natural antibodies produced by the human genome typically contain the CH2 domain of the Fc region, generally via N-linkage. It contains a branched, biantennary oligosaccharide linked to the main Asn297. See, e.g., TIBTECH 15:26-32 (1997). Sugars contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcN Ac), galactose, and sialic acid, as well as Glc in the "stem" of the biantennary oligosaccharide structure In some embodiments, certain improved Modifications of the oligosaccharides in the antibodies of the present application may be carried out to generate antibody variants with improved properties. obtain.

[0223] In some embodiments, the Fc region lacks fucose attached (directly or indirectly). Antibody variants having carbohydrate structures are provided. For example, the amount of fucose in such antibodies is , 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of the source can be determined, for example, by MALDI-T as described in WO2008 / 077546. All sugar structures (e.g., complexes) attached to Asn297 as determined by OF mass spectrometry The total number of glycans at Asn297 (monomeric, hybrid, and high mannose structures) Asn297 is determined by calculating the average amount of fucose in the Fc region. refers to the asparagine residue located at position 297 (EU numbering of Fc region residues), 297 may also be located within approximately ±3 amino acids from position 297 due to minor sequence variations in the antibody. It may be located upstream or downstream, i.e., between positions 294 and 300. Modified variants may have improved ADCC function. See, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyo See "Defucosylation" or "Defucosylation" (Hakko Kogyo Co., Ltd.). Examples of publications relating to "fucose-deficient" antibody variants include US2003 / 0157 108, WO2000 / 61739, WO2001 / 29246, US2003 / 011 5614, US2002 / 0164328, US2004 / 0093621, US200 4 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 0845 70, WO2005 / 035586, WO2005 / 035778, WO2005 / 05 3742, WO2002 / 031140, Okazaki et al.J.Mol.B iol.336:1239-1249(2004), Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing fucosylated antibodies include those deficient in protein fucosylation. Lec13 CHO cells (Ripka et al. Arch. Biochem. B iophys.249:533-545(1986), U.S. Patent Application No. US2003 / 0 No. 157108A1 (Presta, L), and WO2004 / 056312A1 (Ad ams et al., especially Example 11), and the alpha-1,6-fucosyltransferase gene Genes, FUT8, knockout cell lines such as knockout CHO cells (e.g., Yaman e-Ohnuki et al.Biotech.Bioeng.87:614(200 4), Kanda, Y. et al.,Biotechnol.Bioeng.,94( 4):680-688 (2006), and WO2003 / 085107. Examples include:

[0224] For example, biantennary oligosaccharides attached to the Fc region of antibodies are bisected by GlcNAc. Further provided are antibody variants having bisected oligosaccharides containing reduced Examples of such antibody variants include: are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Pat. ,602,684 (Umana et al.), and U.S. Patent No. 2005 / 0123546 (U The oligosaccharides attached to the Fc region contain at least one galactose. Antibody variants with lactic acid residues are also provided. Such antibody variants have improved CDC. Such antibody variants may have a function. et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 ( Raju, S.

[0225] c) Fc region variants In some embodiments, one or more amino acid modifications are made to the Fc The Fc region variant may be one or more A human Fc region sequence (e.g., human Fc region sequence) containing an amino acid modification (e.g., substitution) at the above amino acid position. The antibody may comprise an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0226] In some embodiments, the present application provides a method for the production of antibodies that retain some, but not all, effector functions. Therefore, the in vivo half-life of the antibody is important, but specific effector functions ( We have designed antibody variants that are desirable candidates for applications where other functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays are performed to assess CDC and / or For example, Fc receptor (FcR) binding can be confirmed by the reduction / depletion of ADCC activity. A combination assay was performed to determine whether the antibody lacked FcγR binding (and therefore likely lacked ADCC activity). High FcRn binding capacity can be ensured. NK cells, the primary cells for NK cell proliferation, express only Fc(RIII), whereas monocytes express only Fc(RIII). FcR expression on hematopoietic cells is Ravetch and Kinet,Annu.Rev.Immunol.9:457 The ADCC activity of the molecule of interest is summarized in Table 3 of JP-492 (1991) on page 464. Non-limiting examples of in vitro assays for assessing the activity of hydroxybenzoates are described in U.S. Pat. No. 5,500,366. No. 2 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad Sci. USA 83:7059-7063 (1986)), and He llstrom,I et al.,Proc.Nat'l Acad.Sci.USA 82:1499-1502(1985), 5,821,337(Bruggemann , M. et al., J. Exp. Med. 166: 1351-1361 (1987) Alternatively, non-radioactive assay methods may be used (e.g., For example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Ce llTechnology, Inc.Mountain View, CA, and Cyto Tox 96® non-radioactive cytotoxicity assay (Promega, Madison, MA) Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), PBMCs and natural killer (NK) cells. Additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model, e.g., Clynes et al.Proc.Nat'l Acad.Sci.USA 95: The effects of steroids on the immune system can be evaluated in animal models such as those disclosed in C. C1q binding assay was performed to confirm that the antibody was unable to bind to C1q and thereby inhibit CDC It can also be confirmed that the activity of the compound is lacking. See C1q and C3c binding ELISA in 2005 / 100402. Complement To assess activation, a CDC assay can be performed (e.g., Gazzano-Sa ntoro et al., J. Immunol. Methods 202:163(1 996), Cragg, MSet al., Blood 101:1045-105 2(2003), and Cragg, MS and MJ Glennie, Bloo (See, for example, d 103:2738-2743 (2004)). FcRn binding and In vivo clearance / half-life determinations may be made using methods known in the art. (e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006).

[0227] Antibodies with reduced effector function include those with Fc region residues 238, 265, 26 9, 270, 297, 327, and 329. (U.S. Patent No. 6,737,056). Such Fc variants include those containing amino acid 265. Fc variants with substitutions at two or more of positions 269, 270, 297, and 327. For example, the so-called "DANA" Fc having substitutions of residues 265 and 297 with alanine. Mutants include those described in U.S. Pat. No. 7,332,581.

[0228] Certain antibody variants are described that have improved or reduced binding to FcRs (e.g., , U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001) See .)

[0229] In some embodiments, the antibody variants have one or more amino acid substitutions that improve ADCC. , e.g., at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region The Fc region comprises the following substitutions:

[0230] In some embodiments, for example, U.S. Pat. No. 6,194,551, WO99 / 51 642, and Idusogie et al. J. Immunol. 164:4178- As described in [PubMed], 4184 (2000), the modifications made in the Fc region result in altered Improved (i.e., either improved or reduced) C1q binding and / or complement-dependent cytotoxicity This results in sexual dysfunction (CDC).

[0231] Neonatal Fc receptor (FcRn) is involved in the prolongation of the half-life and transfer of maternal IgG to the fetus Antibodies with improved binding to :587(1976), and Kim et al., J. Immunol. 24:249 (1994)) see US2005 / 0014934A1 (Hinton et al. These antibodies improve the binding of the Fc region to FcRn. Such Fc variants include Fc regions having one or more substitutions therein at Fc region residue 238. , 256, 265, 272, 286, 303, 305, 307, 311, 312, 317 , 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or having substitutions at one or more of 434, e.g., substitutions at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0232] For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40(1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5, See also US Pat. Nos. 624,821 and WO 94 / 29351.

[0233] d) Cysteine ​​Engineered Antibody Variants In some embodiments, the antibody is a cysteine-containing antibody in which one or more residues of the antibody are substituted with a cysteine ​​residue. It may be desirable to generate stain-engineered antibodies, e.g., "thioMabs." In certain embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby introduced into the antibody. and can be used to bind antibodies to other moieties, e.g., drugs. conjugated to a moiety or linker-drug moiety to form an immunomodulatory agent as further described herein. In some embodiments, a conjugate can be made of any of the following residues: Any one or more of the following may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be prepared, for example, by It can be produced as described in US Pat. No. 7,521,541.

[0234] e) Antibody derivatives In some embodiments, the antibodies provided herein are known in the art and and can be further modified to contain additional readily available non-proteinaceous moieties. The moieties suitable for derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol (ethylene glycol), Glycol / propylene glycol copolymer, carboxymethylcellulose, dextran Tolan, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, Poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly( n-Vinylpyrrolidone) Polyethylene Glycol, Propylene Glycol Homopolymer -, propylene oxide / ethylene oxide copolymer, polyoxyethylated poly These include alcohols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Examples include, but are not limited to: Polyethylene glycol propionaldehyde The polymer may be of any molecular weight and may be of any suitable molecular weight. The number of polymers attached to the antibody can vary, and can be 1 or more. If more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or types of polymers used for derivatization will depend on the particular structure of the antibody being improved. properties or functions, whether the antibody derivatives can be used therapeutically under defined conditions, etc. The determination may be based on considerations including, but not limited to:

[0235] In some embodiments, antibodies and non-antibodies that can be selectively heated by exposure to radiation are used. Conjugates of a protein moiety are provided. In some embodiments, a non-protein The active moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad.Sci.USA 102:11600-11605(2005)). Radiation is , a temperature that does not harm normal cells but kills cells in close proximity to the antibody-nonproteinaceous moiety of any wavelength, including but not limited to, wavelengths that heat non-proteinaceous moieties to It can be something.

[0236] Preparation method The antibodies described herein (such as single domain antibodies) may be any antibody known in the art or described herein. It may be prepared using any of the methods described in the literature.

[0237] Methods for preparing single domain antibodies have been described, see, e.g., Els Pardon See et al, Nature Protocols, 2014;9(3):674 Single domain antibodies (VH H, etc.) can be prepared using methods known in the art, e.g. by immunizing a camelid species (such as a camel or llama) and obtaining hybridomas therefrom. or by ligating single domain antibodies using molecular biology techniques known in the art. Then, individual clones of the unselected library were cloned using EL They can be obtained by ISA or by selection using phage display.

[0238] For recombinant production of single domain antibodies, nucleic acid encoding the single domain antibody is isolated. inserted into a replicable vector for further cloning (amplification of the DNA) or expression DNA encoding the single domain antibody can be inserted using conventional techniques (e.g., The oligonucleotides are capable of specifically binding to genes encoding the heavy and light chains of the human body. Many vectors can be easily isolated and sequenced (by using a DNA probe). The choice of vector will depend in part on the host cell to be used. Preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It is of the type.

[0239] 1. Polyclonal antibodies Polyclonal antibodies are generally produced by multiple subcutaneous (sc) or intravenous injections of the relevant antigen and adjuvant. is produced in animals by intraperitoneal (ip) injection. For example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues) ligation), N-hydroxysuccinimide (via lysine residues), glutaraldehyde aldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR, where R and R 1but are independently lower alkyl groups) to produce the relevant antigen in an immunogenic form in the immunizing species. Proteins that are Conjugation to thiamin, bovine thyroglobulin, or soybean trypsin inhibitor Examples of adjuvants that can be used include Freund's complete adjuvant. and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose diphosphate Immunization protocols can be determined by one skilled in the art without undue experimentation. can be selected.

[0240] Animals were administered, for example, 100 μg or 5 μg of protein or conjugate (respectively for rabbits or mice) combined with 3 volumes of Freund's complete adjuvant The antigen, immunogenic conjugate, and or derivatives. One month later, the animals are immunized with 1 / 5 to 1 / 4 of the original volume of peptide or conjugate in complete Freund's adjuvant After 7-14 days, the animals are bled and the serum is assayed for antibody titer. The animals are boosted until the titer plateaus. They can also be produced in recombinant cell culture as protein fusions. The agent is also suitable for enhancing the immune response.

[0241] 2. Monoclonal antibodies Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the antibodies contained in that population The individual antibodies contained herein may contain trace amounts of possible naturally occurring mutations and / or post-translational modifications. They are identical except for modifications (e.g., isomerization, amidation). The modifier "antibody" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0242] For example, monoclonal antibodies were first discovered by Kohler et al., Nature, 256:495 (1975) or may be produced by recombinant DNA methods (US Pat. No. 4,816,567).

[0243] In the hybridoma method, a mouse, or other suitable host animal such as a hamster, is cultured as described above. immunized as described above and capable of specifically binding to the protein used for immunization. Lymphocytes that produce or are capable of producing wax antibodies are induced. Lymphocytes can be immunized in vitro. The lymphocytes are then immunized with polyethylene glycol. and myeloma cells using a suitable fusing agent, such as acetaminophen, to form hybridoma cells. (Goding, Monoclonal Antibodies:Principles and Practice,pp.59-103(Academic Press,1 See 986).

[0244] The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally, humans Peripheral blood lymphocytes ("PBLs") may be used if cells of origin are desired, or non- Whether spleen cells or lymph node cells are used if human mammalian sources are desired. The lymphocytes are then fused using a suitable fusing agent such as polyethylene glycol. It is fused with an immortalized cell line to form a hybridoma cell. lonal Antibodies: Principles and Practice ,Academic Press(1986),pp.59-103.

[0245] Immortalized cell lines are usually derived from transformed mammalian cells, particularly those of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. The prepared hybridoma cells are plated and allowed to grow or differentiate from the unfused parental myeloma cells. are grown in a suitable culture medium that preferably contains one or more substances that inhibit survival. For example, the parent myeloma cells express the enzyme hypoxanthine guanine phosphoribosyltransferase ( In the absence of ATP (HGPRT or HPRT), the culture medium for hybridomas typically contains contains hypoxanthine, aminopterin, and thymidine (HAT medium), which A substance that inhibits the growth of HGPRT-deficient cells.

[0246] Preferred immortalized myeloma cells are those that fuse efficiently and produce stable antibody-producing cells of choice. These support high-level antibody production and are sensitive to media such as HAT medium. Among others, the Salk Institute Cell Distribution MOPC-21 and MOPC-22 available from the National Institute of Physical Therapy, San Diego, Calif. and MPC-11 mouse tumors, USA, and American Type Available from the Culture Collection, Manassas, Va. USA mouse bone marrow cells, such as SP-2 cells (and their derivatives, e.g., X63-Ag8-653), Human myeloma and mouse-human heteromyeloma cell lines are also preferred. The production of human antibodies has been described (Kozbor, J. Immunol., 133: 3001(1984), Brodeur et al., Monoclonal Ant ibody Production Techniques and Applications ions,pp.51-63(Marcel Dekker,Inc.,New York k,1987)).

[0247] The culture medium in which the hybridoma cells grow contains monoclonal antibodies directed against the antigen. Preferably, the antibody is produced by a hybridoma cell. The binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays, e.g., by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). is determined.

[0248] The culture medium in which the hybridoma cells are cultured contains monoclonal antibodies directed against the desired antigen. Preferably, the antibody can be assayed for the presence of a binding parent of a monoclonal antibody. Compatibility and specificity can be determined by immunoprecipitation or in vitro binding assays, e.g., radioimmunoassay. This can be determined by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Techniques and assays for determining binding affinity are known in the art. For example, binding affinity can be determined using the method of Munson et al. et al., Anal. Biochem., 107:220 (1980) This can be determined by code analysis.

[0249] Hybridoma cells that produce antibodies with desired specificity, affinity, and / or activity After identification, clones were subcloned by limiting dilution techniques and analyzed using standard methods. (Goding, supra). Suitable culture media for this purpose include: For example, D-MEM or RPMI-1640 medium can be used. The tumor cells can grow in vivo as tumors in mammals.

[0250] The monoclonal antibodies secreted by the subclones are, for example, protein A-Sepharose. lysis, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography The culture medium, ascites fluid, or or serum.

[0251] Monoclonal antibodies can be prepared by the methods described in U.S. Pat. No. 4,816,567 and the methods described above. Monoclonal antibodies can also be produced by recombinant DNA methods such as those described in The DNA encoding the heavy and light chains of a mouse antibody can be isolated using conventional procedures (e.g., By using oligonucleotide probes that can specifically bind to the gene, ) are readily isolated and sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be used to However, host cells, such as E. coli cells, monkey COS cells, Chinese hamster in ovarian (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. Upon transfection, monoclonal antibodies are synthesized within such recombinant host cells. A review article on the recombinant expression of DNA encoding the ribosomal protein in bacteria is provided by Skerr et al. a et al.,Curr.Opinion in Immunol.,5:256- 262 (1993) and Pliickthun, Immunol. Rev. 130:1 51-188(1992).

[0252] In a further embodiment, the antibody is prepared as described in McCafferty et al., Nature 348:552-554 (1990) They can be isolated from phage libraries. Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. B iol., 222:581-597 (1991), respectively, using phage libraries. This paper describes the isolation of mouse and human antibodies using the ELISA kit. A follow-up report will be published on the isolation of high-affinity antibodies by chain shuffling. Production of human antibodies (nM range) (Marks et al., Bio / Technology y, 10:779-783 (1992)), as well as very large phage libraries. We describe combinatorial infection and in vivo recombination as a strategy to construct Terhouse et al., Nucl. Acids Res., 21:2265- 2266 (1993)). Therefore, these techniques are not suitable for isolating monoclonal antibodies. It is a viable alternative to traditional monoclonal antibody hybridoma technology for

[0253] The DNA may, for example, contain coding sequences for human heavy and light chain constant domains in place of the homologous murine sequences. By substituting sequences (U.S. Pat. No. 4,816,567; Morrison, et al. t al.,Proc.Natl Acad.Sci.USA,81:6851(198 4)), or all or part of the coding sequence for a non-immunoglobulin polypeptide The modification may also be by covalently linking the polypeptide to a globulin coding sequence. Immunoglobulin polypeptides are substituted in place of or incorporated into the constant domains of an antibody. These are substituted for the variable domains of one antigen-binding site of an antibody, One antigen-binding site with specificity for a different antigen and another antigen-binding site with specificity for a different antigen. A chimeric bivalent antibody containing the binding site is generated.

[0254] The monoclonal antibodies described herein may be monovalent, and their preparation may be performed using methods known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at a point within the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue can be replaced with another amino acid residue or cross-linked. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments, specifically Fab fragments, is well known in the art. This can be accomplished using routine techniques known in the art.

[0255] Chimeric or hybrid antibodies are also known in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be prepared in vitro using methods such as disulfide exchange reactions. These can be constructed using reactions or by forming thioether bonds. Suitable reagents for this include iminothiolate and methyl-4-mercaptobutyrimidate. (mercaptobutyrimidate).

[0256] 3. Recombinant production in prokaryotic cells a) Vector construction Polynucleic acid sequences encoding the antibodies of the present application can be obtained using standard recombinant techniques. The desired polynucleic acid sequence is isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be prepared using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide coding sequence can be expressed heterologously in a prokaryotic host. The seed polynucleotide is inserted into a recombinant vector capable of replicating and expressing the gene. Many vectors are available and known in the art and can be used in the present invention. The selection of an appropriate vector depends on the size of the nucleic acid to be inserted into the vector and the size of the host cell to be transformed with the vector. Each vector has its own function (amplification of heterologous polynucleotides) that depends primarily on the specific host cell in which it is used. the extent or expression, or both) and its compatibility with the particular host cell in which it resides. Vector components generally include an origin of replication, a selection marker, and a Car gene, promoter, ribosome binding site (RBS), signal sequence, heterologous nucleic acid insert These sequences include, but are not limited to, transcription entry and termination sequences.

[0257] Generally, a plasmid containing replicon and control sequences derived from a species compatible with the host cell is used. A vector is used in connection with these hosts. The vector typically contains a replication site, and a marking sequence capable of providing phenotypic selection in transformed cells. For example, E. coli typically uses pBR32, a plasmid derived from the E. coli species. pBR322 is transformed using ampicillin (Amp) and tetracycline. It contains a gene encoding cullin (Tet) resistance and therefore can be used to identify transformed cells. pBR322, its derivatives, or other microbial plasmids also provide a convenient means for or bacteriophages are used by microorganisms for the expression of endogenous proteins. The promoter may also contain, or be modified to contain, a promoter that can Examples of pBR322 derivatives used for expression of vectors are described in Carter et al., It is described in detail in US Pat. No. 5,648,237.

[0258] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism. can be used as a transformation vector in connection with these hosts. bacteriophage such as phage 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, The method can be utilized in preparing recombinant vectors that can be used to transform the

[0259] The expression vectors of the present application may contain two or more promoters encoding each of the polypeptide components. The promoter may contain a cistron pair upstream of the cistron whose expression it regulates. 5') of the non-translated regulatory sequence. Prokaryotic promoters typically contain two They are divided into classes, inducible promoters and constitutive promoters. Changes in nutritional conditions, such as the presence or absence of nutrients or changes in temperature, A promoter under which increased levels of transcription of a cistron are initiated.

[0260] A large number of promoters recognized by a variety of potential host cells are well known. The promoter was removed from the source DNA by restriction enzyme digestion and then isolated. By inserting the isolated promoter sequence into the vector of the present application, the light or heavy chain The natural promoter sequence can also be operably linked to the cistron DNA encoding the Either a heterologous promoter can be used to drive amplification and / or expression of the target gene. In some embodiments, the heterologous promoter generally encodes the native target polypeptide promoter. resulting in greater transcription and higher yield of expressed target genes compared to the promoter To achieve this, a heterologous promoter is utilized.

[0261] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, -lac tamase and lactose promoter systems, tryptophan (trp) promoter systems, and and hybrid promoters, such as the tac or trc promoter. However, other promoters functional in bacteria (including other known bacterial or phage promoters) may be used. The nucleic acid sequences of these are publicly available, and therefore, those skilled in the art can easily understand them. may use linkers or adapters to provide any necessary restriction sites. allowing them to be operably linked to the cistrons encoding the target light and heavy chains. (Siebenlist et al. (1980) Cell 20:269).

[0262] In one aspect, each cistron in the recombinant vector is capable of translocating the expressed polypeptide across a membrane. Generally, the signal sequence is a component of the vector. It may be part of the target polypeptide DNA inserted into a vector. The signal sequence selected for clarity should be one that is recognized and processed by the host cell. It should be capable of being cleaved by a signal peptidase. In prokaryotic host cells that do not recognize or process the native signal sequence of the species polypeptide. For example, signal sequences are those of alkaline phosphatase, penicillinase, Ipp , or the heat-stable enterotoxin II (STII) leader, LamB, PhoE, P A prokaryotic signal sequence selected from the group consisting of elB, OmpA, and MBP. In some embodiments of the present application, the cistrons used in both cistrons of the expression system are The signal sequence is the STII signal sequence or a variant thereof.

[0263] In some embodiments, production of antibodies according to the present application may occur in the cytoplasm of the host cell, Therefore, the presence of a secretory signal sequence within each cistron is not required. In the present invention, a polypeptide component, e.g., a first antigen-binding moiety, optionally fused to a second antigen-binding moiety, is V at the joint H A polypeptide encoding a domain and optionally fused to a second antigen-binding moiety. V of the first antigen-binding portion to be L The polypeptides encoding the domains are expressed and folded. Rarely, they assemble to form functional antibodies in the cytoplasm. , E. coli trxB - strain) provides favorable cytoplasmic conditions for disulfide bond formation. thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995) .

[0264] The present invention provides a method for producing a compound that maximizes the yield of secreted and properly assembled antibodies of the present application. The present invention provides an expression system in which the ratio of the expressed polypeptide components can be adjusted. This is achieved at least in part by simultaneously regulating the translational strength of the transcriptional components. One technique for adjusting the intensity is described by Simmons et al., U.S. Pat. No. 5,844,644. No. 0,523, which is a mutation in the translation initiation region (TIR) ​​of a cistron. For a given TIR, there is a range of amino acid or nucleic acid sequence variants. The translation strength of the target gene is then adjusted to the desired expression level of the target gene. TIR variants can be produced by modifying the amino acid sequence. Although it can be generated by conventional mutagenesis techniques that result in codon changes that allow Silent changes are preferred. Modifications of the TIR may include, for example, modifications of the signal sequence, as well as modifications of the signal sequence. This may include alterations in the number or spacing of the C-Dalgarno sequences. One method for generating a signal sequence is to leave the amino acid sequence of the signal sequence unchanged (i.e., The goal is to generate a "codon bank" at the beginning of a coding sequence (where the mutation is silent). This can be achieved by changing the third nucleotide position of each codon, in addition Some amino acids, such as leucine, serine, and arginine, complicate the creation of the bank. This mutagenesis method is described by Yansura et al. al.(1992)METHODS:A Companion to Methods in Enzymol. 4:151-158.

[0265] Preferably, a set of vectors is produced with a range of TIR strengths for each cistron therein. This limited set allows for comparison of the expression levels of each chain, as well as the expression of various TIR intensity sets. The TIR intensity is determined by the Simmons As described in detail in U.S. Pat. No. 5,840,523, the reporter The expression level of the gene can be determined by quantifying the expression level of the gene. Desired individual TIRs are selected to be combined in the expression vector construct of the present application. will be done.

[0266] b) Prokaryotic host cells Suitable prokaryotic host cells for expressing the antibodies of the present application include gram-negative or gram-positive Organisms such as Archaebacteria and Eubacteria are useful. Examples of bacteria include Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis), Enterobacteria, Pseudomonas nas species (e.g. P. aeruginosa), Salmonella typhim urium, Serratia marcescans, Klebsiella, Pro teus, Shigella, Rhizobia, Vitreoscilla, or P In some embodiments, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts in the present invention. An example of an i-strain is strain W3110 (Bachmann, Cellular and Molecular Biology, 2001). Lecular Biology,vol.2(Washington,DC:Am erican Society for Microbiology, 1987), pp. .1190-1219, ATCC accession number 27,325) and its derivatives (genotype W3 110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc-fepE)degP41 kan R strain 33D3, which has (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as Ec oli 294 (ATCC 31,446), E.coli B, E.coli 177 6 (ATCC 31,537) and E. coli RV308 (ATCC 31,608 ) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above bacteria with different genotypes are known in the art. It is known, for example, Bass et al., Proteins, 8:309-314 (1990). In general, the replicability of a replicon in bacterial cells is determined by It is necessary to select an appropriate bacterium taking into consideration the above. For example, pBR322, pBR325 , pACYC177, or pKN410. If provided, E. coli, Serratia, or Salmonella species are the hosts It can be suitably used as

[0267] Typically, the host cell should secrete minimal amounts of proteolytic enzymes and It may be desirable to incorporate suitable protease inhibitors into the cell culture.

[0268] c) Protein production The host cell is transformed with the above-mentioned expression vector, and the promoter is induced, and the transformant is conventional methods modified to be suitable for the selection or amplification of genes encoding desired sequences. Transformation is the process by which DNA is transferred into cells either as an extrachromosomal element or as a chromosomal element. Introduction of DNA into a prokaryotic host so that it can be replicated either by endothelial cells or by Depending on the host cell used, transformation may be carried out using standard techniques appropriate to such cells. Generally, calcium treatment using calcium chloride results in substantial cellular resorption. Another method of transformation is used for bacterial cells that contain a vacuolar barrier. Yet another technique that may be used is electroporation.

[0269] Prokaryotic cells used to produce the antibodies of the present application are known in the art. The cells are grown in a medium suitable for culturing the selected host cells. In some embodiments, the medium may be Luria Broth (LB) containing necessary nutritional supplements. The substrate is a substrate for selectively allowing growth of prokaryotic cells containing the expression vector. The selection agent chosen based on the construction of the vector also contains a selection agent. For example, ampicillin It is added to the medium for growing cells that express the phosphorus resistance gene.

[0270] In addition to sources of carbon, nitrogen, and inorganic phosphate, any necessary supplements may also be added, either singly or in combination. It may be included at an appropriate concentration when introduced as a mixture with another supplement or medium, such as a synthetic nitrogen source. Optionally, the culture medium may contain glutathione, cysteine, cystamine, thioglycolate, One or more hydroxyl groups selected from the group consisting of dithioerythritol, and dithiothreitol It may contain a base agent.

[0271] Prokaryotic host cells are cultured at a suitable temperature. For E. coli growth, for example, a suitable A preferred temperature range is about 20°C to about 39°C, more preferably about 25°C to about 37°C. Even more preferably, the temperature is about 30° C. The pH of the medium ranges from about 5 to about 9, depending mainly on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

[0272] When an inducible promoter is used in the expression vector of the present application, protein expression is In one embodiment of the present application, the PhoA promoter is induced under conditions suitable for the activation of the motor. The gene is used to control the transcription of the polypeptide. is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is C RAP medium (see, e.g., Simmons et al., J. Immunol. (See Methods (2002), 263:133-147). Various other Inducers may be used according to the vector construct used as known in the art.

[0273] The expressed antibodies of the present application are secreted into the periplasm of the host cell and recovered therefrom. Recovery is typically achieved by microfiltration, commonly by means such as osmotic shock, sonication, or lysis. It involves the destruction of the organism. Once the cells are destroyed, the cell debris or whole cells can be separated by centrifugation or filtration. Proteins can be removed by, for example, affinity resin chromatography. Alternatively, the protein may be transported into the culture medium and isolated therein. The cells can be removed from the culture and the culture supernatant can be used for further purification of the produced protein. The expressed polypeptides are analyzed by polyacrylamide gel electrophoresis (PAGE). Further isolation and characterization using commonly known methods such as AGE and Western blot assays and can be identified.

[0274] Alternatively, protein production can be carried out in large quantities by fermentation processes. Fed-batch fermentation techniques are available for the production of recombinant proteins. Large-scale fermentation is at least A capacity of 1000 liters, preferably about 1,000 to 100,000 liters These fermentors provide oxygen and nutrients, especially glucose (the preferred carbon / energy source). A stirring impeller is used to distribute the ghee (source of ghee). Small-scale fermentation is generally is approximately 100 liters or less and is in the range of about 1 liter to about 100 liters This refers to fermentation in a fermentation tank.

[0275] During the fermentation process, induction of protein expression is typically initiated when the cells reach a desired density, e.g. OD of approximately 180-220 550 The stage begins after the plant has grown under favorable conditions until it reaches a At this stage, the cells are in early stationary phase. Various inducers are known in the art and are used as described above. The vector constructs used may be used in accordance with the methods described above. Cells may be grown for a shorter period before induction. Cells are typically induced for approximately 12-50 hours, although longer or shorter induction times can also be used. It can be used.

[0276] Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of the present application. For example, chaperones may be used to improve the proper assembly and folding of secreted polypeptides. Proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl An additional vector overexpressing the cis, trans-isomerase was used to The chaperone protein can be co-transformed in bacterial host cells. It has been demonstrated to facilitate proper folding and solubility of produced heterologous proteins . Chen et al. (1999) J Bio Chem 274:19601-1 9605, Georgiou et al., U.S. Patent No. 6,083,715, Geo rgiou et al., U.S. Patent No. 6,027,888, Bothmann an d Pluckthun(2000)J.Biol.Chem.275:17100-1 7105, Ramm and Pluckthun (2000) J. Biol. Chem. .275:17106-17113, Arie et al. (2001) Mol.Mi crobiol.39:199–210.

[0277] Minimizes proteolysis of expressed heterologous proteins, especially those that are proteolytically sensitive To suppress the production of lactic acid bacteria, certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, the host cell strain may be designed to express known bacterial proteases, such as protease III, Om pT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease Genetic mutation(s) in the gene encoding ATPase VI, and combinations thereof Several E. coli protease-deficient strains are available. For example, see Joly et al. (1998) (see above), Geor giou et al., U.S. Patent No. 5,264,365, Georgiou et al. al., U.S. Patent No. 5,508,192, Hara et al., Microbia l Drug Resistance, 2:63-72(1996).

[0278] A protease-deficient protease-deficient protease that overexpresses one or more chaperone proteins The E. coli strain transformed with the plasmid is used to express the antibody of the present application. can be used as a host cell.

[0279] d) Protein purification The antibodies produced herein may be further purified for further assays and uses. Standard protein purification methods known in the art are used to obtain substantially homogeneous preparations. The following techniques can be used: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation , reverse phase HPLC, chromatography on silica or cation exchange resins such as DEAE, Chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and Sep Gel filtration using hadex G-75 is an example of a suitable purification procedure.

[0280] In one embodiment, Protein A immobilized on a solid phase is an antibody comprising an Fc region of the present application. Protein A is used for affinity purification. It binds to the Fc region of antibodies with high affinity. It is a 41 kD cell wall protein derived from Staphylococcus aureus. Ru. Lindmark et al (1983) J.Immunol.Meth.62: 1-13. The solid phase on which Protein A is immobilized is preferably a glass or silica surface. Preferably, the column is a controlled pore glass column or a silicic acid column. In this application, the column is pre-treated with a reagent such as glycerol to prevent non-specific attachment of contaminants. The solid phase is then washed to remove any non-specifically bound antibodies. Contaminants are removed. Finally, the desired antibody is recovered from the solid phase by elution.

[0281] 4. Recombinant production in eukaryotic cells For eukaryotic expression, vector components generally include a signal sequence, an origin of replication, and a Among the above marker genes, enhancer elements, promoters, and transcription termination sequences, These include, but are not limited to, one or more of:

[0282] a) Signal Sequence Component Vectors for use in eukaryotic hosts may contain a nucleotide sequence encoding the N-terminus of the mature protein or polypeptide. Inserts encoding signal sequences or other polypeptides with specific cleavage sites at the ends The heterologous signal sequence selected preferably is recognized and processed by the host cell. It is digested (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences, as well as viral secretory leaders, e.g. For example, the herpes simplex gD signal can be used.

[0283] The DNA of such precursor region may be a leading sequence for the DNA encoding the antibody of the present application. Ligated in frame.

[0284] b) Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors (although the SV40 origin is typically used). Typically, it may be used only because it contains an early promoter).

[0285] c) Selective Gene Components Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes include (a) antibiotics or other toxins, e.g., ampicillin, conferring resistance to neomycin, methotrexate, or tetracycline (b ) complement an auxotrophic deficiency, or (c) an essential nutrient unavailable from complex media; For example, providing a gene encoding D-alanine racemase for Bacilli Encodes proteins.

[0286] One example of a selection scheme utilizes a drug to arrest growth of the host cell. Successfully transformed cells are transformed with a tandem sequence that confers drug resistance and therefore survives the selection regimen. Examples of such dominant selection include the drugs neomycin, mycophenolic acid, and Use hygromycin.

[0287] Other examples of suitable selectable markers for mammalian cells include DHFR, thymidine kinase, melanoma, and the like. Metallothionein-I and -II, preferably primate metallothionein genes, adenosine Nucleic acids encoding the antibodies of the present application, such as ornithine deaminase and ornithine decarboxylase, are obtained. This allows the identification of cellular components for incorporation.

[0288] For example, cells transformed with the DHFR selection gene are first subjected to a competitive antagonist of DHFR. All transformants were cultured in a medium containing the agonist methotrexate (Mtx). Suitable host cells when wild-type DHFR is used are , Chinese hamster ovary (CHO) cell lines deficient in DHFR activity (e.g., AT CC CRL-9096).

[0289] Alternatively, the polypeptide-encoding DNA sequence, the wild-type DHFR protein, and the amino acid sequence Transgenics with another selectable marker such as glycoside 3'-phosphotransferase (APH) Transformed or co-transformed host cells (especially wild-type hosts containing endogenous DHFR) , aminoglycoside antibiotics such as kanamycin, neomycin, or G418, etc. can be selected by growing cells in medium containing a selection agent for the selectable marker See U.S. Patent No. 4,965,199.

[0290] d) promoter component Expression and cloning vectors are usually vectors that are recognized by the host organism and express the desired polypeptide. It contains a promoter operably linked to a nucleic acid encoding a peptide sequence. All eukaryotic genes are located approximately 25-30 bases upstream from the site where transcription begins. It contains an AT-rich region that is found 70-80 bases upstream from the transcription start of many genes. Another sequence that can be found is the CNCAAT region, where N can be any nucleotide. The 3' end of the organism is a signal for the addition of a polyA tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors. do.

[0291] Other promoters suitable for use with prokaryotic hosts include the phoA promoter, Lactamase and lactose promoter systems, alkaline phosphatase promoters, Tryptophan (trp) promoter systems and hybrid promoters, e.g., t ac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems are operably linked to DNA encoding the antibody. It also contains a Shine-Dalgarno (SD) sequence.

[0292] Transcription of polypeptides from vectors in mammalian host cells can be achieved using, for example, polyomavirus vectors. , fowlpox virus, adenovirus (adenovirus 2, etc.), bovine papillomavirus, chicken meat tumor viruses, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably or from the genome of a virus such as simian virus 40 (SV40), - e.g., from the actin promoter or immunoglobulin promoter, heat shock is controlled by a promoter derived from a promoter, provided that such promoter provided that the vector is compatible with the host cell line.

[0293] The early and late promoters of the SV40 virus also contain the SV40 viral replication origin. The first gene of human cytomegalovirus is conveniently obtained as an SV40 restriction fragment. The bovine promoter is conveniently obtained as a HindIII E restriction fragment. To express DNA in mammalian hosts using papilloma viruses as vectors This system is disclosed in U.S. Pat. No. 4,419,446. Modifications of this system are disclosed in U.S. Pat. ,601,978. Thymidine kinase promoter derived from herpes simplex virus. Regarding the expression of human β-interferon cDNA in mouse cells under the control of the β-interferon gene See also Reyes et al., Nature 297:598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter. do.

[0294] e) enhancer element component Transcription of the DNA encoding the antibody of the present application by higher eukaryotes is often achieved by enzyme The gene expression level of mammalian genes (globin) is increased by inserting an enhancer sequence into the vector. Many enzymes derived from Enhancer sequences are now known, however, typically derived from eukaryotic viruses. For example, the SV40 enhancer on the late side of the replication origin may be used. sensor (bp 100-270), cytomegalovirus early promoter enhancer, Examples include the polyoma enhancer on the late side of the replication origin and the adenovirus enhancer. For enhancing elements for activation of eukaryotic promoters, see Yaniv, Nature See also, e.g., 297:17-18 (1982). Enhancers are polypeptide chains. The promoter sequence may be spliced ​​into the vector at a 5' or 3' position, but is preferably It is located 5' from the target.

[0295] f) transcription termination component Eukaryotic host cells (e.g., yeast, fungi, insect, plant, animal, human, or other multicellular organisms) Expression vectors used in mammalian cells (e.g., mammalian cells) also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences will generally be found in eukaryotic or viral DNA or cDNA. These regions are available from the 5' untranslated region and occasionally the 3' untranslated region of the NA. It is transcribed as a polyadenylated fragment within the untranslated portion of the mRNA encoding the polypeptide. One useful transcription termination component is the bovine growth hormone agonist (BGH) gene. The polyadenylation region is a polyadenylation region. Please refer to the

[0296] g) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include Examples include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. An example of a mammalian host cell line is the SV40 transformed monkey kidney CV1 line (COS -7, ATCC CRL 1651), human embryonic kidney line (293 cells or in suspension culture 293 cells subcloned for growth in Graham et al., JG en Virol. 36:59(1977)); baby hamster kidney cells (BHK, AT CC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Ur aub et al.,Proc.Natl.Acad.Sci.USA 77:421 6 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Repro d.23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587) human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CR L 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (He p G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CC L51);TR1 cells (Mather et al., Annals NYAcad Sci.383:44-68(1982)), MRC5 cells, FS4 cells, and human liver It is a pancreatic cancer strain (Hep G2).

[0297] Host cells are transformed with the above-described expression or cloning vectors for antibody production. , induction of promoters, selection of transformants, or amplification of genes encoding desired sequences. The cells are cultured in conventional nutrient media modified as appropriate for the purpose.

[0298] h) Cultivation of host cells The host cells used to produce the antibody of the present application may be cultured in a variety of media. Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RP MI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Si Commercially available media such as ham e are suitable for culturing host cells. t al., Meth. Enz. 58:44 (1979), Barnes et al. , Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,777 No. 04, No. 4,657,866, No. 4,927,762, No. 4,560,65 No. 5, or No. 5,122,469, WO90 / 03430, WO87 / 0019 5, or any of the media described in U.S. Pat. No. Reissue 30,985 for host cells. Any of these media may optionally contain hormones. and / or other growth factors (such as insulin, transferrin, or epidermal growth factor). , salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (HE PES, etc.), nucleotides (adenosine and thymidine, etc.), antibiotics (GENTAM YCIN™ drugs, trace elements (minerals usually present in minimal concentrations in the micromolar range), Calcium, which is defined as an organic compound, and glucose or an equivalent energy source may be supplemented. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. These culture conditions are not those previously used with the host cell selected for expression. and will be apparent to those skilled in the art.

[0299] i) Protein purification When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or in the culture medium. If antibodies are produced intracellularly, they can be secreted directly. As a first step, they are released into the blood via the particulate debris. (either host cells or lysed fragments) are removed by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) isolated antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is dissolved in sodium acetate (pH 3.5), In the presence of EDTA and phenylmethylsulfonyl fluoride (PMSF), for approximately 30 minutes Cell debris can be removed by centrifugation. Antibodies are secreted into the medium. When used in a protein enrichment system, the supernatant of such an expression system is generally filtered through a commercially available protein concentration filter, e.g. Using an Amicon or Millipore Pellicon ultrafiltration unit Protease inhibitors such as PMSF inhibit proteolysis, The antibiotic may be included in any of the aforementioned steps to inhibit the growth of adventitious contaminants. It may be included to prevent

[0300] Protein compositions prepared from cells can be purified, for example, by hydroxylapatite chromatography. can be purified using chromatography, gel electrophoresis, dialysis, and affinity chromatography; Affinity chromatography is the preferred purification technique. Proteins as affinity ligands The suitability of substance A depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to generate human IgG containing one, two, or four heavy chains. Immunoglobulin-based antibodies can be purified (Lindmark et al. , J. Immunol. Meth. 62:1-13 (1983)). Protein G is a Recommended for all mouse isotypes and human 3 (Guss et al., EMBO J. 5:15671575(1986)). The substrate to which the affinity ligand binds is mostly In this case, the substrate is agarose, but other substrates are available. Controlled pore glass or poly(styrene) Mechanically stable matrices such as ethylene-divinylbenzene can be achieved with agarose. This allows for faster flow rates and shorter processing times than conventional methods. H 3 domains Bakerbond ABX™ resin (JT Baker, Phillips) Purification is accomplished by fractionation on an ion exchange column and ethanol precipitation. Furthermore, reversed-phase HPLC, chromatography on silica, anion or cation exchange resins Heparin SEPHAROSE™ chromatography on a polyaspartic acid column Chromatography on paper, chromatofocusing, SDS-PAGE, and Other techniques for protein purification, such as ammonium sulfate precipitation, may also be used depending on the antibody being recovered. Available.

[0301] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants is purified by: Low pH hydrophobic interaction chromatography using an elution buffer with a pH of approximately 2.5 to 4.5 The method may be carried out at a low salt concentration (e.g., about 0 to 0.25 M salt). .

[0302] Immunoconjugates In some embodiments, the present application provides a method for treating a cancer, comprising administering to a patient a therapeutically effective amount of a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., For example, protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or or fragments thereof), or conjugated to one or more cytotoxic drugs, such as radioisotopes Immunoconjugates comprising any of the antibodies (such as single domain antibodies) described herein. We also offer ugate.

[0303] In some embodiments, the immunoconjugate comprises an antibody that binds to a maytansinoid (US Pat. No. 6,422,162). Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425 235 B1); monomethyl auristatin drug moieties DE and DF (M auristatins such as MAE and MMAF (U.S. Pat. No. 5,635,483 and U.S. Pat. No. 5,635,483); See US Pat. Nos. 5,780,588 and 7,498,298); dolastatins calicheamicin or its derivatives (U.S. Pat. Nos. 5,712,374, 5,712,375, 5,712,376, 5,712,377, 5,712,378, 5,712,379 ... No. 4,586, No. 5,739,116, No. 5,767,285, No. 5,770 ,701, 5,770,710, 5,773,001, and 5,87 No. 7,296, Hinman et al., Cancer Res.53:3336- 3342 (1993), and Lode et al., Cancer Res. 58 :2925-2928 (1998); daunomycin or doxorubicin Anthracyclines (Kratz et al., Current Med. C hem.13:477-523(2006), Jeffrey et al., Bioo rganic & Med.Chem.Letters 16:358-362(200 6), Torgov et al.,Bioconj.Chem.16:717-721 (2005), Nagy et al.,Proc.Natl.Acad.Sci.US A 97:829-834(2000), Dubowchik et al., Bioo rg.& Med.Chem.Letters 12:1529-1532(2002) , King et al., J.Med.Chem.45:4336-4343(200 2) and U.S. Patent No. 6,630,579); methotrexate; docetaxel, paclitaxel, larotaxel, tesetaxel, and orthotaxel Taxanes such as cereals; trichothecenes; and CC1065. Antibody-drug conjugates (ADCs) are antibodies conjugated to one or more drugs.

[0304] In some embodiments, the immunoconjugate comprises diphtheria A chain, diphtheria toxin, The non-binding active fragment of exotoxin A chain (Pseudomonas aeruginosa a), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleur ites fordii protein, diansin protein, Phytolaca am ericana proteins (PAPI, PAPII, and PAP-S), momordi ca charantia inhibitor, curcin, crotin, sapaonaria off icinalis inhibitors, gelonin, mitogenin, restrictocin, phenomycin, enzymatically active toxins, including but not limited to enomycin, and trichothecenes includes an antibody as described herein conjugated to a fragment thereof.

[0305] In some embodiments, the immunoconjugate is conjugated to a radioactive atom. The antibodies described herein may be radioconjugated. can be used for the production of radioactive conjugates. 211 , I 13 1 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , P b 212 Radioactive conjugates are used for detection. When used, it is a radioactive atom for scintigraphy studies, e.g., tc99 m or I123, or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mr Spin labels for i), for example again iodine-123, iodine-131, Indium-111, Fluorine-19, Carbon-13, Nitrogen-15, Oxygen-17, Gadolinium The alloy may contain zinc, manganese, or iron.

[0306] The conjugate of the antibody and the cytotoxic agent may be N-succinimidyl-3-(2-pyridyl)- dithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) ) Cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imino Difunctional derivatives of diesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde ), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), Bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylene diamines), diisocyanates (e.g., toluene 2,6-diisocyanate), and biphenyls. - Active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene) A variety of bifunctional protein binding agents can be used to make, for example, a ricin immunotoxin. Described in Vitetta et al., Science 238:1098 (1987) Carbon-14 labeled 1-isothiocyanatobenzyl Dimethyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a radioactive nucleoside. are exemplary chelating agents for conjugation of oxidases to antibodies. See 1026. The linker facilitates release of the cytotoxic drug inside the cell. It may also be a "cleavable linker." For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (C hari et al., Cancer Res. 52:127-131 (1992), No. 5,208,020) may also be used.

[0307] The immunoconjugate or AD herein C is BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo Ho-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMC C, and sulfo-SMPB, and commercially available (e.g., Pierce Biotec SVSB (from hnology, Inc., Rockford, IL, USA) including, but not limited to, succinimidyl-(4-vinylsulfone)benzoate Such conjugates prepared with crosslinker reagents are expressly contemplated, but are not limited to these. I can't.

[0308] Methods and Compositions for Diagnostics and Detection In some embodiments, among the antibodies provided herein (such as single domain antibodies), Each antibody detects the corresponding antigen (CD19, CD20, BCMA, or CD38) in a biological sample. The term "detecting" as used herein is useful for detecting the presence of In certain embodiments, the biological sample is blood, blood In some embodiments, the biological sample is a cell, a supernatant, or other liquid sample of biological origin. or organization.

[0309] In some embodiments, the anti-CD19 antibodies (of the present invention) for use in diagnostic or detection methods and (e.g., any one of the anti-CD19 single domain antibodies described herein). In another embodiment, a method for detecting the presence of CD19 in a biological sample is provided. In an embodiment, the method comprises detecting the presence of CD19 protein in a biological sample. In certain embodiments, the CD19 is human CD19. The method comprises subjecting a biological sample to the methods described herein under conditions that allow binding of anti-CD19 antibodies to CD19. and contacting the anti-CD19 antibody with the anti-CD19 antibody of the present invention, wherein a complex is formed between the anti-CD19 antibody and CD19. Such methods may be in vitro or in vivo. In some embodiments, the anti-CD19 antibody may be used in combination with anti-CD19 antibody treatment. Used to select eligible subjects, e.g., CD19 is a biomarker for patient selection. It is a marker.

[0310] In some embodiments, the anti-CD20 antibodies (of the present invention) for use in diagnostic or detection methods and (e.g., any one of the anti-CD20 single domain antibodies described herein). In another embodiment, a method for detecting the presence of CD20 in a biological sample is provided. In an embodiment, the method comprises detecting the presence of CD20 protein in a biological sample. In certain embodiments, the CD20 is human CD20. The method comprises subjecting a biological sample to the methods described herein under conditions that allow binding of anti-CD20 antibodies to CD20. and contacting the anti-CD20 antibody with the anti-CD20 antibody of the present invention, wherein a complex is formed between the anti-CD20 antibody and CD20. Such methods may be in vitro or in vivo. In some embodiments, the anti-CD20 antibody may be used in combination with anti-CD20 antibody treatment. Used to select eligible subjects, e.g., CD20 is a biomarker for patient selection. It is a marker.

[0311] In some embodiments, the anti-BCMA antibodies (as disclosed herein) for use in diagnostic or detection methods are and (e.g., any one of the anti-BCMA single domain antibodies described herein). In another embodiment, a method for detecting the presence of BCMA in a biological sample is provided. In an embodiment, the method comprises detecting the presence of BCMA protein in a biological sample. In certain embodiments, the BCMA is human BCMA. The method comprises subjecting a biological sample to the methods described herein under conditions that allow binding of anti-BCMA antibodies to BCMA. and forming a complex between the anti-BCMA antibody and BCMA. Such methods may be in vitro or in vivo. In some embodiments, the anti-BCMA antibody may be administered in a manner consistent with treatment with an anti-BCMA antibody. Used to select eligible subjects, e.g., BCMA is a biomarker for patient selection. It is a marker.

[0312] In some embodiments, an anti-CD38 antibody (the present invention) for use in a method of diagnosis or detection Anti-CD38 single domain antibodies (such as any one of those described herein) are provided. In further embodiments, methods are provided for detecting the presence of CD38 in a biological sample. In embodiments, the method comprises detecting the presence of CD38 protein in a biological sample. In certain embodiments, the CD38 is human CD38. The method comprises subjecting a biological sample to conditions that allow binding of an anti-CD38 antibody to CD38, as described herein. and contacting the anti-CD38 antibody described in claim 1 with the anti-CD38 antibody, wherein the complex is formed between the anti-CD38 antibody and CD38. and detecting whether the protein is formed in the target cell. Such a method may be an in vitro method or an in vivo method. In some embodiments, the anti-CD38 antibody may be administered by treatment with an anti-CD38 antibody. For example, CD38 is used as a biomarker for patient selection. It's Omakar.

[0313] In certain embodiments, a labeled antibody (anti-CD19, anti-CD20, anti-BCMA, or anti- The label may be a directly detectable label or a label that can be used to detect the CD38 single domain antibody. Moieties (fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels, etc.) and enzymes or molecules detected indirectly, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, moieties such as radioactive ions, ligands, and the like. sexual isotope 32 P, 14 C. 125 I, 3 H, and 131 I, fluorophore, e.g., dilute Earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl Lucer, umbelliferone, luceriferase, e.g., firefly lucer luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), arsenic Potassium phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharin Lithooxidases, such as glucose oxidase, galactose oxidase, and Glucose-6-phosphate dehydrogenase, an enzyme that oxidizes pigment precursors using hydrogen peroxide Coupling enzymes (HRP, lactoperoxidase, or microperoxidase, etc.) heterocyclic oxidases, such as uricase and xanthine oxidase, biotin / avidin, spin label, bacteriophage label, stable free radical, etc. However, the present invention is not limited to these.

[0314] III. Chimeric Antigen Receptors Aspects of the present application include one or more single domain antibodies (V H Extracellular antigen-binding domains containing A chimeric antigen receptor (CAR) comprising the single domain antibody described in Section II is provided. Any one of the following may be used in the CARs described herein. H H-do An exemplary CAR containing the main (i.e., V H H-based CARs are shown in Figures 1A-1D. It has been demonstrated that the Those skilled in the art will appreciate that the V in the exemplary CARs of FIGS. H H domain It will be appreciated that may be substituted with other sdAbs.

[0315] In some embodiments, (a) a single domain that specifically binds to an antigen (e.g., a tumor antigen). (b) an extracellular antigen-binding domain containing antibody (sdAb); (c) a transmembrane domain; a chimeric antigen receptor (CAR) comprising a polypeptide comprising an intracellular signaling domain; In some embodiments, the antigen is CD19, CD20, CD22, CD3 3, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, C D138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A 3, and glycolipid F77. In some embodiments, the sdAb is In some embodiments, the transmembrane domain is In the study, CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain is selected from the group consisting of: The main domain contains the primary intracellular signaling domain of immune effector cells (e.g., T cells) In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain is a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises CD27, CD28 , CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, Ligands for LIGHT, NKG2C, B7-H3, CD83, and combinations of these In some embodiments, the CAR is derived from a costimulatory molecule selected from the group consisting of: The hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the CAR further comprises a polypeptide (such as a CD8α hinge domain). A signal peptide (such as CD8α signal peptide) located at the N-terminus of the polypeptide is further In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, the CD8α sequence. signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD28 transmembrane domain main, a costimulatory signaling domain derived from CD28, and a primary domain derived from CD3ζ In some embodiments, the polypeptide comprises an N-terminal intracellular signaling domain. to the C-terminus, CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD28 transmembrane domain, the first costimulatory signaling domain derived from CD28 The second costimulatory signaling domain from CD137 and the second costimulatory signaling domain from CD3ζ In some embodiments, the polypeptide comprises a primary intracellular signaling domain comprising: From N-terminus to C-terminus, CD8α signal peptide, extracellular antigen-binding domain, CD8α The hinge domain, CD8α transmembrane domain, and costimulatory signaling domain derived from CD137 It contains the primary intracellular signaling domain derived from CD3ζ and the CD3ζ domain. In some embodiments, the CAR is monospecific. In some embodiments, the CAR is multivalent, such as bivalent or trivalent. In embodiments, the CAR is multispecific, such as bispecific.

[0316] Specific targeting of chimeric antigen receptors In some embodiments, the present application relates to the anti-CD19, anti-CD20, anti-B an extracellular antigen binding antibody, including either CMA, or an anti-CD38 single domain antibody CARs containing domains are provided. CARs can be monospecific or multispecific (bispecific). or higher specificity, etc.), and the CAR can be monovalent or multivalent (bivalent, trivalent, or Exemplary monospecific chimeric antigen receptors, exemplary sequences: A list of the constructs and their vectors is shown in Table 4.

[0317] Tables 4, 5, and 6 listed in section "III. Chimeric Antigen Receptors" use the following abbreviations: Ex.: exemplary, Vec.: vector, AA: amino acid sequence of CAR, NA: C AR nucleic acid sequence, SP: signal peptide, extracellular: extracellular antigen-binding domain, sdAb : Single domain antibody, TM: Transmembrane domain, CO1: Costimulatory signaling domain 1, CO2: costimulatory signaling domain 2, Prim.: primary intracellular signaling domain The domains correspond to the order of the domains from the N-terminus to the C-terminus of the CAR polypeptide. They are listed from left to right on each line.

[0318] 1.CD19 CAR In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD19 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. CD19-targeting CARs (also referred to herein as "CD19 CARs") containing In some embodiments, the anti-CD19 sdAb is a camelid, chimeric, In some embodiments, the intracellular signaling domain is: Contains the primary intracellular signaling domains of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the CD19 CAR is derived from a costimulatory molecule selected from the group consisting of: The hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the CD19 C AR contains a signal peptide (CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises, from N-terminus to C-terminus: CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD2 8 transmembrane domains, a costimulatory signaling domain derived from CD28, and a CD3ζ-derived In some embodiments, the CD19 C The AR is monospecific. In some embodiments, the CD19 CAR is monovalent. In some embodiments, the CD19 CAR is multispecific, such as bispecific. In some embodiments, the CD19 CAR is multivalent, such as bivalent or trivalent.

[0319] In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD19 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. and a CD19 CAR comprising the amino acid sequence of SEQ ID NO: 1. CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 In some embodiments, the anti-CD19 sdAb comprises a camelid, chimeric , human, or humanized. In some embodiments, the anti-CD19 sdAb has the sequence FR1 comprising the amino acid sequence of SEQ ID NO: 240, FR2 comprising the amino acid sequence of SEQ ID NO: 241, FR3 comprising the amino acid sequence of SEQ ID NO: 242 and / or the amino acid sequence of SEQ ID NO: 243 In some embodiments, the anti-CD19 sdAb further comprises an FR4 comprising the sequence V containing amino acid sequence number 76 H In some embodiments, the intracellular The signaling domain is responsible for primary intracellular signaling in immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain comprises a CD3 In some embodiments, the intracellular signaling domain is derived from a costimulatory signal. In some embodiments, the costimulatory signaling domain comprises a CD4+ transduction domain. 27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, C D2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands, and In some embodiments, the co-stimulatory molecule is selected from the group consisting of a combination of: The CD19 CAR is a fusion protein consisting of the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. It also contains a hinge domain (such as the CD8α hinge domain) located between the two. In embodiments, the CD19 CAR comprises a signal peptide ( In some embodiments, the polypeptide further comprises a CD8α signal peptide. , from N-terminus to C-terminus, CD8α signal peptide, extracellular antigen-binding domain, CD8 α-hinge domain, CD28 transmembrane domain, costimulatory signaling domain derived from CD28 It contains the primary intracellular signaling domain derived from CD3ζ and the CD3ζ domain. In some embodiments, the CD19 CAR is monospecific. 9 CAR is monovalent. In some embodiments, the CD19 CAR is bispecific. In some embodiments, the CD19 CAR is bivalent or trivalent. and other polyvalent compounds.

[0320] In some embodiments, the amino acid sequence of SEQ ID NO: 248 is at least about 85%, 86%, or 90% identical to the amino acid sequence of SEQ ID NO: 248. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity In some embodiments, a CD19 CAR is provided that comprises a polypeptide having SEQ ID NO: A CD19 CAR comprising the amino acid sequence of SEQ ID NO: 248 is provided. Polypeptides comprising the amino acid sequences are also provided.

[0321] In some embodiments, any of the CD19 CARs provided herein In some embodiments, an isolated nucleic acid encoding the nucleic acid of SEQ ID NO: 250 is provided. Acid sequence and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, an isolated nucleic acid is provided that has sequence identity to any one of: In some embodiments, an isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 250 is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the CD19 CAR is a CD4-encoded RNA. In some embodiments, a vector is provided that comprises any one of the nucleic acids. In some embodiments, the vector is a lentivirus. vectors, such as viral vectors.

[0322] 2.CD20 CAR In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD20 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. CD20-targeted CARs (also referred to herein as "CD20 CARs") containing In some embodiments, the anti-CD20 sdAb is a camelid, chimeric, In some embodiments, the intracellular signaling domain is: Contains the primary intracellular signaling domains of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the CD20 CAR is derived from a costimulatory molecule selected from the group consisting of: The hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the CD20 C AR contains a signal peptide (CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises, from N-terminus to C-terminus: CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD2 8 transmembrane domains, a costimulatory signaling domain derived from CD28, and a CD3ζ-derived In some embodiments, the CD20 C The AR is monospecific. In some embodiments, the CD20 CAR is monovalent. In some embodiments, the CD20 CAR comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, the CAR is multispecific, such as bispecific. In embodiments, the CD20 CAR is multivalent, such as bivalent or trivalent.

[0323] In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD20 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. and a CD20 CAR comprising the amino acid sequence of SEQ ID NO: 4. CDR2 comprising the amino acid sequence of SEQ ID NO:5, and the amino acid sequence of SEQ ID NO:6 In some embodiments, the anti-CD20 sdAb is a camelid, chimeric , human, or humanized. In some embodiments, the anti-CD20 sdAb has the sequence FR1 comprising the amino acid sequence of SEQ ID NO: 244, FR2 comprising the amino acid sequence of SEQ ID NO: 245, FR3 comprising the amino acid sequence of SEQ ID NO: 246 and / or the amino acid sequence of SEQ ID NO: 247 In some embodiments, the anti-CD20 sdAb further comprises a FR4 comprising the sequence V containing amino acid sequence number 77 H In some embodiments, the intracellular The signaling domain is responsible for primary intracellular signaling in immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain comprises a CD3 In some embodiments, the intracellular signaling domain is derived from a costimulatory signal. In some embodiments, the costimulatory signaling domain comprises a CD4+ transduction domain. 27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, C D2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands, and In some embodiments, the co-stimulatory molecule is selected from the group consisting of a combination of: CD20 CAR is a CD20 CAR consisting of a C-terminal extracellular antigen-binding domain and an N-terminal transmembrane domain. It also contains a hinge domain (such as the CD8α hinge domain) located between the two. In embodiments, the CD20 CAR comprises a signal peptide ( In some embodiments, the polypeptide further comprises a CD8α signal peptide. , from N-terminus to C-terminus, CD8α signal peptide, extracellular antigen-binding domain, CD8 α-hinge domain, CD28 transmembrane domain, costimulatory signaling domain derived from CD28 It contains the primary intracellular signaling domain derived from CD3ζ and the CD3ζ domain. In some embodiments, the CD20 CAR is monospecific. In some embodiments, the CD20 CAR is monovalent. In some embodiments, the CAR comprises a multispecific, such as bispecific, sequence of 49 amino acids. In some embodiments, the CD20 CAR is multivalent, such as bivalent or trivalent. be.

[0324] In some embodiments, the amino acid sequence of SEQ ID NO: 249 is at least about 85%, 86%, or 90% identical to the amino acid sequence of SEQ ID NO: 249. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity In some embodiments, a CD20 CAR is provided that comprises a polypeptide having SEQ ID NO: A CD20 CAR comprising the amino acid sequence of SEQ ID NO: 249 is provided. Polypeptides comprising the amino acid sequences are also provided.

[0325] In some embodiments, any of the CD20 CARs provided herein In some embodiments, an isolated nucleic acid encoding the nucleic acid of SEQ ID NO: 251 is provided. Acid sequence and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, an isolated nucleic acid is provided that has sequence identity to any one of: In some embodiments, an isolated nucleic acid is provided comprising the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the CD20 CAR is a gene encoding the CD20 CAR described above. In some embodiments, a vector is provided that comprises any one of the nucleic acids. In some embodiments, the vector is a lentivirus. vectors, such as viral vectors.

[0326] 3.BCMA CAR In some embodiments, (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. BCMA-targeting CARs (also referred to herein as "BCMA CARs") containing In some embodiments, the anti-BCMA sdAb is a camelid, chimeric, In some embodiments, the intracellular signaling domain is: Contains the primary intracellular signaling domains of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the BCMA CAR is derived from a costimulatory molecule selected from the group consisting of: The hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the BCMA C domain further comprises a CD8α hinge domain. AR contains a signal peptide (CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises, from N-terminus to C-terminus: CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD2 8 transmembrane domains, first costimulatory signaling domain derived from CD28, CD137 a second costimulatory signaling domain derived from CD3ζ, and a primary intracellular signaling domain derived from CD3ζ In some embodiments, the polypeptide comprises an N-terminal to C-terminal CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, The CD8α transmembrane domain, the costimulatory signaling domain derived from CD137, and the CD In some embodiments, the primary intracellular signaling domain is derived from BC 3ζ. The BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monospecific. It is a price.

[0327] In some embodiments, (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. The BCMA CAR is provided comprising an anti-BCMA sdAb, which is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 18 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 29; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 19 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 30; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 20 DR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 31; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 10, and CDR2 comprising the amino acid sequence of SEQ ID NO: 21 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 32; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 22 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 33; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 12 and the amino acid sequence of SEQ ID NO: 23 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 34; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 24 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 25 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 36; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 26 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 37; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, and CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38; or (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 17, and CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. include. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or human. In some embodiments, the anti-BCMA sdAb is selected from SEQ ID NOs: 78-88. V containing an amino acid sequence from the group consisting of H In some embodiments, the cell The intracellular signaling domain is the primary intracellular signaling domain for immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain comprises a C In some embodiments, the intracellular signaling domain is derived from a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1 , CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and In some embodiments, the co-stimulatory molecule is selected from the group consisting of: BCMA CARs are composed of the C-terminal extracellular antigen-binding domain and the N-terminal transmembrane domain. and further contain a hinge domain (such as the CD8α hinge domain) located between them. In this embodiment, the BCMA CAR comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises a signal peptide (such as a CD8α signal peptide). The domain consists of, from the N-terminus to the C-terminus, the CD8α signal peptide, the extracellular antigen-binding domain, and the C D8α hinge domain, CD28 transmembrane domain, the first costimulatory signal derived from CD28 a null transduction domain, a second costimulatory signaling domain derived from CD137, and a CD In some embodiments, the primary intracellular signaling domain is derived from poly(A)-3ζ. The peptide consists of, from the N-terminus to the C-terminus, the CD8α signal peptide, the extracellular antigen-binding domain, and The CD8α hinge domain, CD8α transmembrane domain, and costimulatory signaling domain derived from CD137 It contains a signal transduction domain and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the BCMA CAR is monospecific. So, BCMA CAR is univalent.

[0328] In some embodiments, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 152-162 and 257-259 A nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, or 100% amino acid sequence identity with the selected amino acid sequence. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and BCMA C polypeptides containing 100% or more of a sequence identity to any one of In some embodiments, SEQ ID NOs: 152-162 and 257-25 are provided. 9. A BCMA CAR is provided comprising an amino acid sequence selected from the group consisting of SEQ ID NO: A polynucleotide comprising an amino acid sequence selected from the group consisting of Nos. 152-162 and 257-259. Peptides are also provided.

[0329] In some embodiments, any of the BCMA CARs provided herein In some embodiments, an isolated nucleic acid encoding SEQ ID NOs: 175-176 is provided. 85 and 261-263 and at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity In some embodiments, isolated nucleic acids are provided that are selected from the group consisting of SEQ ID NOs: 175-185 and An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of 261-263 is provided. In some embodiments, the isolated nucleic acid is DNA. The isolated nucleic acid is RNA. In some embodiments, the isolated nucleic acid is a BCMA nucleic acid as described above. Vectors containing any one of the nucleic acids encoding a CAR are provided. In embodiments, the vector is an expression vector. In some embodiments, the vector is and viral vectors such as lentiviral vectors.

[0330] 4.CD38 CAR In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD38 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. CD38-targeted CARs (also referred to herein as "CD38 CARs") containing In some embodiments, the anti-CD38 sdAb is a camelid, chimeric, In some embodiments, the intracellular signaling domain is: Contains the primary intracellular signaling domains of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the CD38 CAR is derived from a costimulatory molecule selected from the group consisting of: The hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the CD38 C AR contains a signal peptide (CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises, from N-terminus to C-terminus: CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD2 8 transmembrane domains, first costimulatory signaling domain derived from CD28, CD137 a second costimulatory signaling domain derived from CD3ζ, and a primary intracellular signaling domain derived from CD3ζ In some embodiments, the polypeptide comprises an N-terminal to C-terminal CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, The CD8α transmembrane domain, the costimulatory signaling domain derived from CD137, and the CD In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the CD38 CAR is monospecific. It is a price.

[0331] In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD38 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. The CD38 CAR is provided, comprising an anti-CD38 sdAb, which is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, and CDR2 comprising the amino acid sequence of SEQ ID NO: 52 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 64; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 41, and CDR2 comprising the amino acid sequence of SEQ ID NO: 53 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 42, and CDR2 comprising the amino acid sequence of SEQ ID NO: 54 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO: 55 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 67; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, and CDR2 comprising the amino acid sequence of SEQ ID NO: 56 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 45 and the amino acid sequence of SEQ ID NO: 57 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 69; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 58 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 70; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 47, and CDR2 comprising the amino acid sequence of SEQ ID NO: 59 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 48, and CDR2 comprising the amino acid sequence of SEQ ID NO: 60 CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 49, and CDR2 comprising the amino acid sequence of SEQ ID NO: 61 and CDR3 comprising the amino acid sequence of SEQ ID NO: 73; (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 50, and CDR2 comprising the amino acid sequence of SEQ ID NO: 62 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74; or (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 51, and CDR2 comprising the amino acid sequence of SEQ ID NO: 63 and CDR3 comprising the amino acid sequence of SEQ ID NO: 75. include. In some embodiments, the anti-CD38 sdAb is camelid, chimeric, human, or humanized. In some embodiments, the anti-CD38 sdAb is selected from the group consisting of SEQ ID NOs: 89-100. V comprising an amino acid sequence from the group consisting of H In some embodiments, the H domain comprises: The intracellular signaling domain is the primary intracellular signaling domain of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain comprises a In some embodiments, the intracellular signaling domain is derived from CD3ζ. In some embodiments, the costimulatory signaling domain comprises a , CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA- 1, ligands for CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83; and The costimulatory molecule is derived from a costimulatory molecule selected from the group consisting of these combinations. In this state, the CD38 CAR is bounded by the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. It further contains a hinge domain (such as the CD8α hinge domain) located between the ends. In some embodiments, the CD38 CAR comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the polypeptide further comprises a signal peptide (such as a CD8α signal peptide). The CD8α signal peptide, the extracellular antigen-binding domain, and the CD8α signal peptide are contained in the CD8α protein, from the N-terminus to the C-terminus. CD8α hinge domain, CD28 transmembrane domain, the first costimulatory signaling domain derived from CD28 a signal transduction domain, a second costimulatory signaling domain derived from CD137, and a C In some embodiments, the primary intracellular signaling domain is derived from poly(D3ζ). The polypeptide consists of, from the N-terminus to the C-terminus, the CD8α signal peptide, the extracellular antigen-binding domain, In, CD8α hinge domain, CD8α transmembrane domain, costimulatory domain derived from CD137 signaling domain, and the primary intracellular signaling domain derived from CD3ζ In some embodiments, the CD38 CAR is monospecific. In this state, the CD38 CAR is monovalent.

[0332] In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 163-174 and 260. 85%, 86%, 87%, 88%, 89%, 90%, 91%, or more of the amino acid sequence %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % of the sequence identity of a CD38 CAR. In some embodiments, a sequence selected from the group consisting of SEQ ID NOs: 163-174 and 260 is provided. CD38 CARs comprising selected amino acid sequences are provided. SEQ ID NOs: 163-174 and 260.

[0333] In some embodiments, any of the CD38 CARs provided herein In some embodiments, an isolated nucleic acid encoding SEQ ID NOs: 186-189 is provided. 97 and 264 and at least about 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% sequence identity. In some embodiments, nucleic acids selected from SEQ ID NOs: 186-197 and 264 are provided. An isolated nucleic acid is provided comprising a nucleic acid sequence selected from the group consisting of: In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the CD38 CAR is a CD38 CAR encoding the CD38 CAR described above. In some embodiments, a vector is provided that comprises any one of the nucleic acids. In some embodiments, the vector is a lentivirus. vectors, such as viral vectors.

[0334] 5.CD22 CAR In some embodiments, (a) an extracellular antigen-binding domain comprising an anti-CD22 sdAb (b) a transmembrane domain; and (c) an intracellular signaling domain. CD22-targeted CARs (also referred to herein as "CD22 CARs") containing In some embodiments, the anti-CD22 sdAb is a camelid, chimeric, In some embodiments, the intracellular signaling domain is: Contains the primary intracellular signaling domains of immune effector cells (e.g., T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD13 7, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT , NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the CD22CAR is derived from a costimulatory molecule selected from the group consisting of: A hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain In some embodiments, the CD22 CA R is a signal peptide located at the N-terminus of the polypeptide (e.g., CD8α signal peptide). In some embodiments, the polypeptide further comprises, from N-terminus to C-terminus, C D8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD28 Transmembrane domain, the first costimulatory signaling domain derived from CD28, CD137 a second costimulatory signaling domain derived from CD3ζ, and a primary intracellular signaling domain derived from CD3ζ In some embodiments, the polypeptide comprises an N-terminal to C-terminal domain. CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, C D8α transmembrane domain, costimulatory signaling domain derived from CD137, and CD3 In some embodiments, the primary intracellular signaling domain is derived from CD2 2. The CAR is monospecific. In some embodiments, the CD22 CAR is monovalent. is. [Table 4-1] [Table 4-2]

[0335] Multivalent Chimeric Antigen Receptor The present application provides two or more (about 2, 3, 4, 5, 6, or more) antibodies, including single domain antibodies. Also provided are multivalent CARs having antigen binding sites of any one of the following: In embodiments, a multivalent CAR targets a single antigen and has two binding sites for a single antigen. In some embodiments, a multivalent CAR targets two or more antigens and includes a multivalent C The AR contains two or more binding sites for at least one antigen. The binding sites may bind to the same epitope of the antigen or may bind to different epitopes of the antigen. Binding sites specific for the same antigen may comprise the same or different single domain antibodies. .

[0336] In some embodiments, the present application provides (a) a compound that specifically binds to an antigen (such as a tumor antigen). a number (such as about any one of 2, 3, 4, 5, 6, or more) of single domain antibodies; (b) an extracellular antigen-binding domain containing antibody (sdAb); (c) a transmembrane domain; and Multivalent (bivalent, trivalent, or higher) polypeptides comprising an intracellular signaling domain and In some embodiments, the antigen is a chimeric antigen receptor (e.g., CD19). , CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, N Y-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the plurality of sdAbs are camelid, chimeric, human, or humanized. In some embodiments, the single domain antibodies are peptide-linked or peptide-phosphorylated. In some embodiments, each peptide linker is about 5 A value of 0 or less (approximately 35, 25, 20, 15, 10, or 5 or less, etc.) In some embodiments, the transmembrane domain is a CD8α, CD4, CD5α, or CD6α amino acid length. 28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain is In some embodiments, the primary intracellular signaling domain comprises a primary intracellular signaling domain (e.g., a T cell). The intracellular signaling domain is derived from CD3ζ. In some embodiments, the signaling domain comprises a costimulatory signaling domain. , costimulatory signaling domains are CD27, CD28, CD137, OX40, CD3 0, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7- H3, a ligand for CD83, and a combination thereof. In some embodiments, the multivalent CAR is derived from the C-terminus of the extracellular antigen binding domain. The hinge domain (CD8α hinge domain) is located between the end of the CD8α and the N-terminus of the transmembrane domain. In some embodiments, the multivalent CAR further comprises a polypeptide at the N-terminus thereof. In some embodiments, the signal peptide further comprises a signal peptide (such as the CD8α signal peptide) that locates the target gene. In this form, the polypeptide comprises, from the N-terminus to the C-terminus, the CD8α signal peptide, the extracellular Antigen-binding domain, CD8α hinge domain, CD8α transmembrane domain, CD137-derived The costimulatory signaling domain derived from CD3ζ and the primary intracellular signaling domain derived from CD3ζ In some embodiments, the multivalent CAR is monospecific. In embodiments, the multivalent CAR is multispecific, such as bispecific.

[0337] In some embodiments, the present application provides a method for detecting a first epitope of an antigen (e.g., a tumor antigen) comprising: (a) detecting a first epitope of an antigen (e.g., a tumor antigen); A first single domain antibody that specifically binds to a second epitope of the antigen and a second single domain antibody that specifically binds to a second epitope of the antigen. (b) an extracellular antigen-binding domain comprising a second single domain antibody; and (b) a transmembrane domain; (c) a multivalent (bivalent, trivalent, or or higher valency, etc.), and In some embodiments, the antigens are CD19, CD20, CD22, CD 33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the first s The dAb and / or the second sdAb may be camelid, chimeric, human or humanized. In some embodiments, the first single domain antibody and the second single domain antibody are peptides. In some embodiments, the nucleotides are fused to each other via a peptide bond or a peptide linker. The peptide linker may be about 50 or less (e.g., about 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the transmembrane domain is , CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and P In some embodiments, the intracellular signaling domain is selected from the group consisting of: D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D3 contains the primary intracellular signaling domain of immune effector cells (such as T cells). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD 137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIG Ligands for HT, NKG2C, B7-H3, CD83, and combinations thereof In some embodiments, the multivalent CAR is derived from a costimulatory molecule selected from the group A hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain (such as a CD8α hinge domain). In some embodiments, the multivalent CAR further comprises: The signal peptide located at the N-terminus of t...

Claims

1. An anti-CD38 single domain antibody (sdAb) selected from the group consisting of SEQ ID NOs: 89-100. CDR1, CDR2 defined by an sdAb containing any one of the amino acid sequences, and Anti-CD38 sdAb containing CDR3.

2. wherein the anti-CD38 sdAb is selected from the group of sdAbs comprising any of the following: The anti-CD38 sdAb of claim 1: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, and CDR2 comprising the amino acid sequence of SEQ ID NO: 52 a CDR2 comprising the amino acid sequence of SEQ ID NO: 64; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 41, and CDR2 comprising the amino acid sequence of SEQ ID NO: 53 a CDR2 comprising the amino acid sequence of SEQ ID NO: 65; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 42, and CDR2 comprising the amino acid sequence of SEQ ID NO: 54 a CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 43, and CDR2 comprising the amino acid sequence of SEQ ID NO: 55 a CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 67; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, and CDR2 comprising the amino acid sequence of SEQ ID NO: 56 a CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 45, and CDR2 comprising the amino acid sequence of SEQ ID NO: 57 a CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 69; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 46, and CDR2 comprising the amino acid sequence of SEQ ID NO: 58 a CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 70; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 47, and CDR2 comprising the amino acid sequence of SEQ ID NO: 59 a CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 48, and CDR2 comprising the amino acid sequence of SEQ ID NO: 60 a CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 49, the amino acid sequence of SEQ ID NO: 61 a CDR2 comprising the amino acid sequence of SEQ ID NO: 73; (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 50, the amino acid sequence of SEQ ID NO: 62 a CDR2 comprising the amino acid sequence of SEQ ID NO: 74; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74; or (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 51, the amino acid sequence of SEQ ID NO: 63 and CDR3 comprising the amino acid sequence of SEQ ID NO:

75.

3. The anti-CD38 sdAb comprises the amino acid sequence of any one of SEQ ID NOs: 89-100.

3. The anti-CD38 sdAb of claim 1 or 2, comprising the sequence:

4. (a) an extracellular antigen-binding antibody comprising the anti-CD38 sdAb of any one of claims 1 to 3; a combined domain; (b) a transmembrane domain; and (c) a multivalent chimeric antigen receptor comprising a polypeptide comprising an intracellular signaling domain. Condition (CAR).

5. The anti-CD38 sdAb is any one of SEQ ID NOs: 92-94 and 97-100 CDR1, CDR2, and CDR3 defined by an sdAb containing one or more amino acid sequences The CAR of claim 4, comprising R3.

6. wherein the anti-CD38 sdAb is selected from the group of sdAbs comprising any of the following: The CAR according to claim 5: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 43, and CDR2 comprising the amino acid sequence of SEQ ID NO: 55 a CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 67; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, and CDR2 comprising the amino acid sequence of SEQ ID NO: 56 a CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 45, and CDR2 comprising the amino acid sequence of SEQ ID NO: 57 a CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 69; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 48, and CDR2 comprising the amino acid sequence of SEQ ID NO: 60 a CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 49, and CDR2 comprising the amino acid sequence of SEQ ID NO: 61 a CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 73; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 50, and CDR2 comprising the amino acid sequence of SEQ ID NO: 62 a CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74; or (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 51, and CDR2 comprising the amino acid sequence of SEQ ID NO: 63 and a CDR3 comprising the amino acid sequence of SEQ ID NO:

75.

7. The anti-CD38 sdAb is any one of SEQ ID NOs: 92-94 and 97-100 The CAR according to claim 5, comprising one or more amino acid sequences.

8. The anti-CD38 sdAb is a camelid, chimeric, humanized, or human antibody. The CAR according to any one of claims 4 to 7.

9. (a) a first sdAb that specifically binds to a first antigen and a second sdAb that specifically binds to a second antigen; and a second sdAb comprising the antibody of any one of claims 1 to 3. an extracellular antigen-binding domain that is a CD38 sdAb; and (b) a transmembrane domain; and (c) a polypeptide comprising an intracellular signaling domain.

10. The first antigen is different from the second antigen, or the first antigen is different from the second antigen. The CAR of claim 9, which is the same as the antigen.

11. The first sdAb is located at the N-terminus of the second sdAb, or The CAR of claim 9 or 10, which is located at the C-terminus of the dAb.

12. The second antigen is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, E from GFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77 The CAR according to any one of claims 9 to 11, selected from the group consisting of:

13. wherein the second sdAb is an anti-BCMA sdAb or an anti-CD38 sdAb. The CAR according to claim 12.

14. The first sdAb is defined by an sdAb comprising the amino acid sequence of SEQ ID NO:

93. 9 to 1, which is an anti-CD38 sdAb comprising CDR1, CDR2, and CDR3.

4. The CAR according to any one of claims 3 to 3.

15. the anti-CD38 sdAb comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 44; CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO:

68. The CAR of claim 14, wherein the sdAb comprises

16. 15. The method of claim 14, wherein the anti-CD38 sdAb comprises the amino acid sequence of SEQ ID NO:

93. CAR.

17. The second sdAb is defined by an sdAb comprising the amino acid sequence of SEQ ID NOs: 78-88. and wherein the antibody is an anti-BCMA sdAb comprising CDR1, CDR2, and CDR3 as defined above.

17. The CAR according to any one of 13 to 16.

18. The second sdAb is an anti-CD38 sdAb according to any one of claims 1 to 3. The CAR according to any one of claims 13 to 16.

19. The second sdAb is defined by an sdAb comprising the amino acid sequence of SEQ ID NO:

93.

19. The method of claim 18, wherein the antibody is an anti-CD38 sdAb comprising CDR1, CDR2, and CDR3. The CAR described herein.

20. the anti-CD38 sdAb comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 44; CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO:

68. The CAR of claim 19, wherein the CAR is an sdAb comprising

21. 20. The method of claim 19, wherein the anti-CD38 sdAb comprises the amino acid sequence of SEQ ID NO:

93. CAR.

22. The first sdAb and the second sdAb may target the same epitope or different epitopes. The CAR according to claim 9 or 10, which specifically binds to a topope.

23. the first sdAb and / or the second sdAb are Camelid, chimeric, humanized, The CAR according to any one of claims 9 to 23, which is an antibody or a human antibody.

24. The first anti-CD38 sdAb and the second anti-CD38 sdAb are peptide-bound. fused to each other via a peptide linker having a length of 50 amino acids or less.

24. The CAR according to any one of 23.

25. The transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, and CD8 6, CD152, and PD1, according to claims 4 to 24. The CAR according to any one of the preceding claims.

26. The intracellular signaling domain is a primary intracellular signaling domain of an immune effector cell. domain, wherein the primary intracellular signaling domain is derived from CD3ζ.

26. The CAR according to any one of claims 1 to 25.

27. The intracellular signaling domain is selected from the group consisting of CD27, CD28, CD137, OX40, C D30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B Co-stimulatory agents selected from the group consisting of 7-H3, a ligand for CD83, and combinations thereof.

27. The method of claim 4, comprising a co-stimulatory signaling domain derived from a stimulatory molecule. The CAR described herein.

28. A hinge located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain Main, and Any of claims 4 to 27, further comprising a signal peptide located at the N-terminus of the polypeptide. The CAR according to any one of the preceding claims.

29. A method for producing a nucleic acid sequence comprising the steps of: The CAR according to claim 4 or 9.

30. An isolated antibody comprising a nucleic acid sequence encoding the CAR according to any one of claims 4 to 29. Nucleic acid.

31. A vector comprising the isolated nucleic acid of claim 30.

32. The engineered immune effector cells are sd cells according to any one of claims 1 to 3. Ab, the CAR according to any one of claims 4 to 29, and the isolated nucleus according to claim 30. or the vector of claim 31, The engineered immune effector cell, wherein the immune effector cell is a T cell.

33. The sdAb according to any one of claims 1 to 3, the sdAb according to any one of claims 4 to 29 30. The isolated nucleic acid of claim 31, the vector of claim 32, or the isolated nucleic acid of claim 33.

33. A method for producing a vaccine comprising administering to a subject the engineered immune effector cells of claim 32 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:

34. 10. The method of claim 1, wherein the compound is used to prepare a pharmaceutical composition for treating cancer in an individual. sdAb according to any one of claims 1 to 3, and CAR according to any one of claims 4 to 29; Or the engineered immune effector cell of claim 32.

35. In the treatment of cancer in an individual, comprising administering to the individual an effective amount of said pharmaceutical composition.

34. The pharmaceutical composition of claim 33 for use in

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