CN117083292A

CN117083292A - CS1 engineered cells and compositions thereof

Info

Publication number: CN117083292A
Application number: CN202280022824.1A
Authority: CN
Inventors: 李宗海; 王鹏
Original assignee: Keji Biomedical Shanghai Co ltd
Current assignee: Keji Biomedical Shanghai Co ltd
Priority date: 2021-04-02
Filing date: 2022-04-01
Publication date: 2023-11-17
Also published as: WO2022206941A1

Abstract

An antigen binding unit capable of targeting CS1 and uses thereof are provided. To immunoconjugates, chimeric receptors comprising said CS 1-targeting antigen binding units, nucleic acids encoding said CS 1-targeting antigen binding units. Also relates to the use of said CS 1-targeting antigen binding units, immunoconjugates, chimeric receptors, host cells for the treatment of diseases.

Description

CS1 engineered cells and compositions thereof

RELATED APPLICATIONS

The present application claims priority from the chinese application No. 202110363275.1 filed on 2/4/2021.

Technical Field

The application relates to an antigen binding unit capable of targeting CS1 protein and application thereof.

Background

Multiple Myeloma (MM) is a cancer of plasma cells in bone marrow, and in general, plasma cells produce antibodies and play a key role in immune function. However, uncontrolled growth of these cells leads to bone pain and fractures, anemia, infection, and other complications. Although the exact cause of multiple myeloma is not known, multiple myeloma is the second most common hematological malignancy, accounting for 2% of all cancer deaths. MM is a heterogeneous disease and is mostly caused by chromosomal translocations of t (11; 14), t (4; 14), t (8; 14), del (13), del (17) (among others) (Drach et al, (1998) Blood 92 (3): 802-809; gertz et al, (2005) Blood 106 (8): 2837-2840; facon et al, (2001) Blood 97 (6): 1566-1571). The main pathology of Multiple Myeloma (MM) is the unlimited expansion and enrichment of plasma cells in the bone marrow, which in turn leads to osteonecrosis. MM affected patients may experience a variety of symptoms associated with disease due to psychological burden of bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and cancer diagnosis. The main treatment scheme at present is chemotherapy and stem cell transplantation, wherein the chemotherapy drugs are mainly steroids, thalidomide, lenalidomide, bortezomib or a combination of a plurality of cytotoxin reagents, and the high-dose chemotherapy concept can be adopted for younger patients to match with autologous stem cell transplantation.

CS1 is a lymphocyte signaling activating molecule (seventh member of SLAM family, CD319 antigen), a member of the CD2 family of cell surface glycoproteins. Based on the high expression of CS1 in multiple myeloma, it is used as a target for the preparation of antibodies or CAR-T cells for the treatment of multiple myeloma and CS 1-positive tumors.

Disclosure of Invention

The application aims to provide an antigen binding unit of anti-CS 1 capable of specifically inhibiting the growth of human multiple myeloma and other CS1 positive tumor cells, and T immune cells (CAR-T) expressing chimeric antigen receptor containing the antigen binding unit sequence, which play a role in tumor adoptive immunotherapy.

The technical scheme provided by the application comprises the following steps:

in a first aspect, there is provided an antigen binding unit targeting CS1, the antigen binding unit being selected from the group consisting of:

(1) An antigen binding unit comprising a heavy chain variable region comprising HCDR1 as shown in SEQ ID No. 3, 13 or 23, and/or comprising HCDR2 as shown in SEQ ID No. 4, 14, 24 or 32, and/or comprising HCDR3 as shown in SEQ ID No. 5, 15, 25 or 41; (2) An antigen binding unit comprising a light chain variable region comprising LCDR1 as set forth in SEQ ID No. 8, 18 or 28, and/or comprising LCDR2 as set forth in SEQ ID No. 9, 19 or 29, and/or comprising LCDR3 as set forth in SEQ ID No. 10, 20 or 30; (3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit; (4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antigen binding unit of any one of (1) to (3).

In a specific embodiment, the antigen binding unit is selected from the group consisting of: (1) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 4, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; or (2) an antigen binding unit comprising HCDR1 shown in SEQ ID NO. 13, HCDR2 shown in SEQ ID NO. 14, HCDR3 shown in SEQ ID NO. 15, LCDR1 shown in SEQ ID NO. 18, LCDR2 shown in SEQ ID NO. 19, LCDR3 shown in SEQ ID NO. 20; or (3) an antigen binding unit comprising HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 25, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30; or (4) an antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; or (5) an antigen binding unit comprising HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 41, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30; (6) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (5), having the same or similar activity as the antigen binding unit of any one of (1) to (5).

In a specific embodiment, the antigen binding unit is selected from the group consisting of:

(1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO: 1. 11, 21, 31, 36 or 40; (2) An antigen binding unit, said antigen binding unit having a light chain variable region having the amino acid sequence of SEQ ID NO: 6. 16, 26, 34, 38 or 43; (3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit; (4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antibody of any one of (1) to (3).

(1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:6, an amino acid sequence shown in figure 6; (2) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:11 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no; (3) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no; (4) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no; (5) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:38, and a nucleotide sequence shown in seq id no; (6) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:43, an amino acid sequence shown in seq id no; (7) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (6), having the same or similar activity as the antigen binding unit of any one of (1) to (6).

In a specific embodiment, the antigen binding unit recognizes the same epitope as any of the antigen binding units described above; or Ig-like V-type domain binding CS1 protein; or Ig-like C2-type domain binding CS1 protein.

In a specific embodiment, the antigen binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody, or a fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is a fully anti-scFv, fv fragment, fab 'fragment, (Fab') ₂ Fragments, fd fragments, dAb fragments, single domain antibodies, multifunctional antibodies or scFv-Fc antibodies.

A second aspect provides an immunogenic conjugate, the immunogenic conjugate comprising: an antigen binding unit according to the first aspect; and functional molecules attached thereto.

A third aspect provides a chimeric receptor whose extracellular domain comprises the antigen binding unit of the first aspect, the chimeric receptor comprising: chimeric Antigen Receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), or a combination thereof.

In a specific embodiment, the chimeric receptor comprises, in sequential linkage: an antigen binding unit, a transmembrane region and an intracellular signaling region of the first aspect.

In a specific embodiment, the intracellular signaling region of the chimeric receptor is selected from the group consisting of: CD3 ζ, fceriγ, CD27, CD28, CD137, CD134, myD88, intracellular signal region sequences of CD40, or combinations thereof; and/or the transmembrane region comprises a transmembrane region of CD8 or CD 28.

In a specific embodiment, the chimeric receptor comprises: an antigen binding unit of the first aspect, a transmembrane region of CD8/CD28, and cd3ζ; or the antigen binding unit of the first aspect, the transmembrane region of CD8/CD28, the intracellular signaling region of CD137, and cd3ζ; or the antigen binding unit of the first aspect, the transmembrane region of CD8/CD28, the intracellular signaling region of CD28, and cd3ζ; or the antigen binding unit of the first aspect, the transmembrane region of CD8/CD28, the intracellular signaling region of CD28, CD137 and CD3 ζ.

In a specific embodiment, the chimeric receptor has the amino acid sequence set forth in SEQ ID NO: 45. 46 or 47.

A fourth aspect provides a nucleic acid encoding the antigen binding unit of the first aspect, the immunoconjugate of the second aspect, the chimeric receptor of the third aspect.

In a fifth aspect there is provided an expression vector comprising a nucleic acid as described in the fourth aspect.

A sixth aspect provides a virus comprising the expression vector of the fifth aspect.

A seventh aspect provides a composition comprising an antigen binding unit according to the first aspect, an immunoconjugate according to the second aspect, and/or a chimeric receptor according to the third aspect, characterized in that the composition is cytotoxic to cells expressing CS 1.

In a specific embodiment, the CS1 expressing cells are tumor cells and/or pathogen cells.

In an eighth aspect, there is provided a host cell comprising an expression vector according to the fifth aspect or a nucleic acid according to the fourth aspect integrated into the genome.

In a specific embodiment, the host cell expresses the chimeric receptor of the third aspect.

In a specific embodiment, the host cell comprises a T cell, a cytotoxic T lymphocyte, an NK cell, a NKT cell, a DNT cell, a regulatory T cell, an NK92 cell, a stem cell derived immune effector cell, or a combination thereof.

In a specific embodiment, the T cell is a T cell derived from a natural T cell and/or induced by a pluripotent stem cell; preferably, the T cells are autologous or allogeneic T cells; preferably, the T cell is a primary T cell; preferably, the T cells are derived from autologous human T cells.

In a specific embodiment, the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), αβ T cells, γδ T cells, or a combination thereof.

In a specific embodiment, the host cell comprises: the knockout of the gene encoding the TCR protein and/or the endogenous TCR molecule is expressed or not, and/or the knockout of the gene encoding the MHC protein and/or the endogenous MHC is expressed or not.

In a specific embodiment, the host cell knocks out endogenous MHC molecule B2M and endogenous TCR using CRISPR/Cas9 technology.

In a specific embodiment, the host cell knocks out B2M using a gRNA comprising the sequences set forth in SEQ ID NOS 84, 85, 86 and/or 87, and the host cell knocks out TCR using a gRNA comprising the sequences set forth in SEQ ID NOS 76, 77, 78, 79, 80, 81, 82 and/or 83.

In a specific embodiment, the host cell comprises a knockout of the gene encoding the CS1 protein and/or low or no expression of the endogenous CS1 molecule.

In a specific embodiment, the host cell CS1 gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the sequences set forth in SEQ ID NOs 88, 89, 90, 91, 92, 93, 94 and/or 95.

In a specific embodiment, the host cell binds to cells that express CS1 without significantly binding to cells that do not express CS 1.

In a specific embodiment, the host cell further carries a coding sequence for an exogenous cytokine; or it also expresses another chimeric receptor; or it also expresses chemokine receptors; or it also expresses a safety switch.

A ninth aspect provides for the administration of a combination of an antigen binding unit according to the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a composition according to the seventh aspect, a host cell according to the eighth aspect, in combination with an agent that enhances its function, preferably in combination with a chemotherapeutic agent; and/or in combination with an agent that ameliorates one or more side effects associated therewith; and/or in combination with host cells expressing chimeric receptors targeted beyond CS 1.

A tenth aspect provides a method of preparing an antigen binding unit according to the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, and/or a composition according to the seventh aspect, the method comprising culturing a host cell according to the eighth aspect under conditions suitable for expression of the antigen binding unit, immunoconjugate, chimeric receptor, and isolating the antigen binding unit, immunoconjugate, chimeric receptor, and/or composition expressed by the host cell.

An eleventh aspect provides a pharmaceutical composition comprising: an antigen binding unit according to the first aspect or a nucleic acid encoding the antigen binding unit; or the immunological conjugate of the second aspect or a nucleic acid encoding the conjugate; or the chimeric receptor of the third aspect or a nucleic acid encoding the chimeric receptor; or the host cell of the eighth aspect; and optionally, a pharmaceutically acceptable carrier or excipient.

A twelfth aspect provides a method of treating/diagnosing a disease comprising administering to a subject in need thereof an effective amount of an antigen binding unit as described in the first aspect, or an immunoconjugate as described in the second aspect, or a host cell as described in the third aspect, or a composition as described in the seventh aspect; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; preferably the tumour is multiple myeloma; preferably, the subject is a human; preferably, wherein the host cell is an autologous or allogeneic T cell to the subject.

A thirteenth aspect provides the use of an antigen binding unit according to the first aspect, or an immunoconjugate according to the second aspect, or a host cell according to the eighth aspect, or a composition according to the seventh aspect, for the treatment and/or diagnosis of a disease, characterized in that the disease expresses CS1; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors, preferably the tumor is multiple myeloma.

A fourteenth aspect provides the use of an antigen binding unit as described in the first aspect, or an immunoconjugate as described in the second aspect, or a host cell as described in the eighth aspect, or a composition as described in the seventh aspect, for the manufacture of a medicament for killing NK cells.

In a specific embodiment, the use increases the persistence and/or the survival rate of transplantation of autologous or allogeneic immune cells in the presence of host immune cells.

The application also relates to: in one aspect, the application provides a CS 1-targeted antigen binding unit selected from the group consisting of: (1) An antigen binding unit comprising a heavy chain variable region comprising HCDR1 as shown in SEQ ID No. 3, 13 or 23, and/or comprising HCDR2 as shown in SEQ ID No. 4, 14, 24 or 32, and/or comprising HCDR3 as shown in any one of SEQ ID nos. 5, 15, 25 or 41; (2) An antigen binding unit comprising a light chain variable region comprising LCDR1 as set forth in SEQ ID NOs 8, 18, 28, and/or comprising LCDR2 as set forth in SEQ ID NOs 9, 19, 29, and/or comprising LCDR3 as set forth in any one of SEQ ID NOs 10, 20 or 30; (3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit; (4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antigen binding unit of any one of (1) to (3).

In specific embodiments, the antigen binding unit is selected from the group consisting of: (1) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 4, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; (2) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 13, HCDR2 shown in SEQ ID NO. 14, HCDR3 shown in SEQ ID NO. 15, LCDR1 shown in SEQ ID NO. 18, LCDR2 shown in SEQ ID NO. 19, LCDR3 shown in SEQ ID NO. 20; (3) An antigen binding unit, HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 25, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30; (4) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; or (5) an antigen binding unit comprising HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 41, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30; (6) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (5), having the same or similar activity as the antigen binding unit of any one of (1) to (5).

In specific embodiments, the antigen binding unit is selected from the group consisting of: (1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO: 11. 11, 21, 31, 36 or 40; (2) An antigen binding unit, said antigen binding unit having a light chain variable region having the amino acid sequence of SEQ ID NO: 6. 16, 26, 34, 38 or 43; (3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit; (4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antigen binding unit of any one of (1) to (3).

In specific embodiments, the antigen binding unit is selected from the group consisting of: (1) An antigen binding unit, the heavy chain variable region of said antibody having the amino acid sequence of SEQ ID NO:1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:6, an amino acid sequence shown in figure 6; (2) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:11 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no; (3) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no; (4) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no; (5) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:38, and a nucleotide sequence shown in seq id no; (6) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:43, an amino acid sequence shown in seq id no; (7) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (6), having the same or similar activity as the antigen binding unit of any one of (1) to (6).

In one aspect, the application provides an antigen binding unit that recognizes the same epitope as the antigen binding unit of the application; or Ig-like V-type domain binding CS1 protein; or Ig-like C2-type domain binding CS1 protein.

In specific embodiments, the antigen binding unit is a humanized antibody, chimeric antibody, or fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is scFv, fv, fab or (Fab) ₂ 。

In a specific embodiment, the antigen binding unit is a humanized antibody selected from the group consisting of: (1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no; (2) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:38, and a nucleotide sequence shown in seq id no; (3) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:43, and an amino acid sequence shown in SEQ ID NO. 43

In one aspect, the application provides nucleic acids encoding antibodies of the application.

In one aspect, the application provides an expression vector comprising a nucleic acid according to the application.

In one aspect, the application provides a host cell comprising an expression vector or genome according to the application, into which a nucleic acid according to the application has been integrated.

In one aspect, the application provides an antigen binding unit of an antibody of the application for use in the preparation of a targeting drug, antibody drug conjugate or multifunctional antibody that specifically targets CS1 tumor cells; or (b)

For preparing a reagent for diagnosing a tumor, the tumor expressing CS1; or for preparing chimeric antigen receptor-modified immune cells; preferably, the immune cell comprises: t lymphocytes, NK cells or NKT lymphocytes.

In one aspect, the application provides a multifunctional immunogenic conjugate comprising:

an antigen binding unit according to the application; and a functional molecule attached thereto; the functional molecule is selected from the group consisting of: molecules targeting tumor surface markers, molecules inhibiting tumors, molecules targeting surface markers of immune cells or detectable markers.

In a specific embodiment, the tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin, preferably the cytokine comprises: IL-12, IL-15, type I interferon, TNF-alpha.

In specific embodiments, the molecule that targets a surface marker of an immune cell is an antibody or ligand that binds to a surface marker of an immune cell; preferably, the immune cell surface marker comprises: preferably, the antibody that binds to an immune cell surface marker is an anti-CD 3 antibody, CD3, CD16, CD28,4-1 BB.

In specific embodiments, the molecule that targets a surface marker of an immune cell is an antibody that binds a T cell surface marker.

In one aspect, the application provides a nucleic acid encoding a multifunctional immunoconjugate of the application.

In one aspect, the application provides the use of a multifunctional immunological conjugate according to the application for the preparation of an anti-tumour agent, or for the preparation of a reagent for diagnosing a tumour expressing CS1; or for preparing chimeric antigen receptor-modified immune cells; preferably, the immune cell comprises: t lymphocytes, NK cells or NKT lymphocytes.

In one aspect, the application provides a chimeric antigen receptor comprising an extracellular domain comprising an antibody according to the application, preferably a single chain antibody or domain antibody, a transmembrane domain and an intracellular signaling domain.

In particular embodiments, the intracellular signal domain comprises one or more co-stimulatory signal domains and/or primary signal domains.

In particular embodiments, the chimeric antigen receptor further comprises a hinge domain.

In specific embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, zeta chain, CD3 epsilon, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the transmembrane region of PD 1; and/or the costimulatory signaling domain is selected from the group consisting of the intracellular signaling regions of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, fceriy, myD88, and 41 BBL; and/or the primary signal domain is selected from tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d, and CD3 ζ, more preferably the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD45, PD1, CD154 and CD 28; and/or the costimulatory signal domain is selected from the group consisting of CD137, CD134, CD28 and OX40; and/or the primary signal domain is selected from cd3ζ, optimally, the transmembrane domain is selected from CD8 a or CD28, the costimulatory signal domain is selected from the intracellular signal domain of CD137 or CD28, and the primary signal domain is selected from cd3ζ.

In a specific embodiment, the chimeric antigen receptor comprises an antigen binding unit, a transmembrane region and an intracellular signaling region linked in that order: the antigen binding unit, the transmembrane region of CD8 and CD3 zeta; the antigen binding unit, the transmembrane region of CD8, the intracellular signal region of CD137 and CD3 zeta; the antigen binding unit, the transmembrane region of CD28, the intracellular signal region of CD28 and CD3 zeta; or an antigen binding unit according to the application, a transmembrane region of CD28, an intracellular signaling region of CD28, CD137 and CD3 ζ.

In a specific embodiment, said extracellular domain of said chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO. 50, 51 or 52; the transmembrane domain is selected from the group consisting of SEQ ID NO:54, a CD8 transmembrane domain shown as seq id NO:72, a CD28 transmembrane domain; the costimulatory signal domain is selected from the group consisting of SEQ ID NOs: 73, the CD28 intracellular domain shown in SEQ ID NO:56 and the CD3 zeta intracellular signaling domain shown in SEQ ID NO:55 or a mixture thereof.

In specific embodiments, the chimeric antigen receptor is selected from the group consisting of: chimeric antigen receptor one having the extracellular domain shown in SEQ ID NO. 50, SEQ ID NO:53 and the hinge domain shown in seq id NO:54, the transmembrane domain shown in SEQ ID NO:55, and the co-stimulatory signaling domain shown in SEQ ID NO:56 (hu 32a12 BBz); or chimeric antigen receptor one having the extracellular domain shown in SEQ ID NO:51, SEQ ID NO:53 and the hinge domain shown in seq id NO:54, the transmembrane domain shown in SEQ ID NO:55, and the co-stimulatory signaling domain shown in SEQ ID NO:56 (hu 37A3 BBz); or chimeric antigen receptor one having the extracellular domain shown in SEQ ID NO:52, SEQ ID NO:53 and the hinge domain shown in seq id NO:54, the transmembrane domain shown in SEQ ID NO:55, and the co-stimulatory signaling domain shown in SEQ ID NO:56 (hu 48G9 BBz).

In one aspect, the application provides nucleic acids encoding the chimeric antigen receptors described herein.

In one aspect, the application provides a virus comprising a vector of the application.

In a preferred embodiment, the virus is a lentivirus.

In one aspect, the application provides the use of a chimeric antigen receptor according to the application, or a nucleic acid according to the application, or an expression vector according to the application, or a virus according to the application, for the preparation of a genetically modified immune cell targeting a CS1 expressing tumor cell,

in a preferred embodiment, the CS1 expressing tumor is multiple myeloma.

In one aspect, the application provides a genetically modified immune cell transduced with a nucleic acid of the application, or an expression vector of the application or a virus of the application; or expressed with the chimeric antigen receptor of the application,

the immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.

In specific embodiments, the genetically modified immune cell is further expressed with other sequences in addition to the chimeric antigen receptor of the application, including a cytokine, or another chimeric antigen receptor, or a chemokine receptor, or an siRNA that reduces expression of PD-1, or a protein that blocks PD-L1, or a TCR, or a safety switch; preferably, the cytokine comprises IL-12, IL-15, IL-21, or type I interferon; preferably, the chemokine receptor comprises CCR2, CCR5, CXCR2, or CXCR4; preferably, the safety switch comprises icaspas-9, purified EGFR or RQR8.

In one aspect, the application provides the use of a genetically modified immune cell according to the application for the preparation of a medicament for inhibiting a tumor, wherein the tumor is a CS1 expressing tumor, and in a preferred embodiment, the CS1 expressing tumor is multiple myeloma.

In one aspect, the present application provides a pharmaceutical composition comprising: the antibody or nucleic acid encoding the antibody of the application; or an immunological conjugate of the application or a nucleic acid encoding the conjugate; or a chimeric antigen receptor of the application or a nucleic acid encoding the chimeric antigen receptor; or genetically modified immune cells according to the application.

In one aspect, the present application provides a kit comprising: a container, and a pharmaceutical composition of the application in the container; or a container, and an antibody of the application or a nucleic acid encoding the antibody in the container; or an immunological conjugate of the application or a nucleic acid encoding the conjugate; or a chimeric antigen receptor of the application or a nucleic acid encoding the chimeric antigen receptor; or genetically modified immune cells according to the application.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the binding of CS1 hybridoma antibodies 32A12MAb, 37A3MAb, 48G9MAb to recombinant protein hSLAMF7-avi-His as determined by ELISA.

FIG. 2 shows binding of CS1 hybridoma antibody to MM.1S, a multiple myeloma cell line.

FIG. 3 shows binding of CS1 hybridoma antibodies to human, murine, and monkey 3 species of SLAMF7 recombinant proteins, respectively.

FIG. 4 shows binding of CS1 hybridoma antibodies to MM.1S, NCI H929, RPMI 8226 and WI38, HEK293 cells.

FIG. 5 shows the binding of humanized CS1 antibodies hu32A12, hu37A3, hu48G9 to recombinant protein hSLAMF 7-avi-His.

FIG. 6 shows the binding of humanized CS1 antibodies to multiple myeloma cell lines.

FIG. 7 shows the binding of humanized CS1 antibodies to human, murine, and monkey 3 species SLAMF7 recombinant proteins, respectively.

FIG. 8 shows the binding of humanized CS1 antibody to MM.1S, NCI H929 and WI38, HEK293 cells.

FIG. 9 shows the affinity assay of humanized CS1 antibody hu37A3 with human SLAMF 7.

Fig. 10 shows the affinity assay of humanized CS1 antibody hu48G9 with human SLAMF 7.

FIG. 11 shows the affinity assay of humanized CS1 antibody huLuc63 with human SLAMF 7.

FIG. 12 shows the affinity assay of the CS1 antibody Luc90 with human SLAMF 7.

FIG. 13 shows the affinity assay of humanized CS1 antibody hu37A3 with monkey SLAMF 7.

FIG. 14 shows the affinity assay of humanized CS1 antibody hu48G9 with monkey SLAMF 7.

Fig. 15 shows the affinity assay of humanized CS1 antibody hu32a12 with human SLAMF 7.

Fig. 16 shows the aggregation results of humanized CS1 antibody hu37A3 from SEC assay.

Fig. 17 shows the aggregation results of the humanized CS1 antibody hu32a12 by SEC assay.

Fig. 18 shows the aggregation results of humanized CS1 antibody hu48G9 from SEC assay.

Figure 19 shows the positive rate of CS1 CAR T cell CARs.

Figure 20 shows the positive rate of CS1 CAR T cell CARs at different time points.

Figure 21 shows in vitro killing results of CS1 CAR T against CS1 expressing positive and negative cells at different potency target ratios.

FIG. 22 shows IFN- γ secretion following co-incubation of CS1 CAR T with CS1 expressing positive and negative target cells.

FIG. 23 shows TNF- α secretion following co-incubation of CS1 CAR T with CS1 expressing positive and negative target cells.

FIG. 24 shows IL-2 secretion following co-incubation of CS1 CAR T with CS1 expressing positive and negative target cells.

Figure 25 shows the effect of soluble CS1 on CS1 CAR T cell killing in vitro.

FIG. 26 shows expression of CS1 CAR T cells PD-1 following stimulation by CS 1-positive multiple myeloma cells.

FIG. 27 shows expression of CS1 CAR T cells Tim-3 following stimulation with CS 1-positive multiple myeloma cells.

FIG. 28 shows expression of CS1 CAR T cells LAG-3 following stimulation by CS 1-positive multiple myeloma cells.

FIG. 29 shows the western blot results of CS1 CAR T cell phosphorylating CD3- ζ (CAR).

FIG. 30 shows IL-6 secretion following co-incubation of CS1 CAR T cells with monocytes and CS1 positive multiple myeloma cells.

Figure 31 shows in vitro expansion of CS1 CAR T cells after stimulation with target cells.

Figure 32 shows the viability of CS1 CAR T cells at various time points after stimulation with target cells.

FIG. 33 shows in vitro amplification and viability of UTD following stimulation by target cells.

FIG. 34 shows in vitro expansion and viability of CS1 CAR T cells under IL-2 stimulation.

Figure 35 shows a comparison of tumor photographs over time in a CS1 CAR T cell in vivo treatment multiple myeloma NPG mice subcutaneous engraftment tumor model.

FIG. 36 shows the tumor-inhibiting and anti-tumor effects of CS1 CAR T cells on human multiple myeloma cell RPMI-8226-CS1 in NPG mice subcutaneously.

FIG. 37 shows the anti-tumor effect of CS1-UCAR-T cells, CS1-UCAR-CS1-/-T cells on NPG mice subcutaneously transplanted with human multiple myeloma cells RPMI 8226-CS 1.

Detailed Description

The inventors have conducted extensive and intensive studies and have unexpectedly found antigen binding units that specifically bind to CS1, which can be used for the preparation of various targeted antitumor drugs and drugs for diagnosing tumors. The present application has been completed on the basis of this finding.

Terminology

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise specified.

The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are well explained in the literature. See, e.g., current Protocols in Molecular Biology (FrederickM.AUSUBEL, 2000,Wileyand sonInc,Library of Congress,USA); molecular Cloning: A Laboratory Manual, third Edition, (sambrook et al,2001,Cold Spring Harbor,NewYork:Cold Spring Harbor Laboratory Press); oligonucleotide Synthesis (m.j. Gaited., 1984); mullis et al U.S. Pat.No.4,683,195; nucleic Acid Hybridization (B.D.Harries & S.J.Higginseds.1984); transcription And Translation (B.D.Hames & S.J.Higginseds.1984); culture Of Animal Cells (r.i. freshney, alan r.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); perbal, A Practical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m.simon, eds. -in-coef, academic Press, inc., new York), especially vols.154 and 155 (wuetal.eds.) and vol.185, "Gene Expression Technology" (d. Goeddel, ed.); gene Transfer Vectors For Mammalian Cells (j.h.miller and M.P.Caloseds.,1987,Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., academic Press, london, 1987); hand book Of Experimental Immunology, volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1986).

In the disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is understood that each intervening value, to the extent that it is between the upper and lower limit of that range and any other stated or intervening value in that range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where a range includes one or both of the limits, the claimed subject matter also includes ranges excluding either or both of those limits. This applies regardless of the width of the range.

The term "about" as used herein refers to the usual error range for each value as readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X". For example, "about" or "comprising" may mean within 1 or more than 1 according to the actual standard deviation in the field. Or "about" or "comprising" may mean a range of up to 10% (i.e., ±10%). For example, about 5 μm may include any number between 4.5 μm and 5.5 μm.

Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range recited herein is to be understood as including any integer within the range, and where appropriate, fractions thereof (e.g., tenths and hundredths of integers) of numerical values.

For a better understanding of the application, the relevant terms are defined as follows:

the term "CS1" (also known as SLAMF7, CD319 or CRACC-NCBI reference sequence: NP-067004.3) is a lymphocyte activating molecule family member 7 that is involved in cell adhesion and NK cell activation functions, and is primarily expressed in plasma cells, NK cells, CD8+ T cells, activated B cells and mononuclear dendritic cells, but is not substantially expressed in progenitor cells of the hematopoietic lineage and other tissues of the human body. CS1 is a type I transmembrane protein, the extracellular segment consisting of serine (S) -methionine (M) at position 23, which contains the two domains Ig-like V region (23 serine S-124 valine V, distal) and Ig-like C region (131 proline P-206 serine S, proximal). Exemplary, the amino acid sequence of the extracellular portion of human SLAMF7 is set forth in SEQ ID NO:57, the amino acid sequence of the extracellular domain of murine SLAMF7 is set forth in SEQ ID NO:58, the amino acid sequence of the cynomolgus monkey SLAMF7 extracellular domain is set forth in SEQ ID NO: 59. CS1 molecules are highly expressed in Multiple Myeloma (MM) cells, and a monoclonal antibody of the CS1 molecules, namely, the ELotuzumab (huLuc 63), has good clinical results in treating patients with relapsed or refractory MM by combining an immunomodulator and a proteasome inhibitor, and has been approved by the FDA for treating MM.

The hybridoma antibody for recognizing CS1 and the humanized antibody modified by the hybridoma antibody can be used for treating multiple myeloma. The CAR-T cells containing the CS1 antibody prepared by the application can obviously kill multiple myeloma cells in vitro and in vivo. Because CS1 is expressed on NK cells, CAR-T cells comprising the application recognizing CS1 antibodies are resistant to killing of autologous or allogeneic T cells by NK cells in a host, thereby increasing the persistence and/or engraftment survival of autologous or allogeneic immune cells (e.g., CS1-CAR-T cells, CS1-UCAR-CS1-/-T cells) in the presence of host immune cells. The bispecific antibody comprising the CS1 antibody prepared by the application and the recognition T cell antibody can resist the killing of autologous or allogeneic T cells by NK cells in a host, thereby increasing the persistence and/or the transplantation survival rate of autologous or allogeneic immune cells (exemplified CS1-CAR-T cells, CS1-UCAR-CS1-/-T cells) in the presence of the host immune cells.

The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, cyclic or branched, it may comprise modified amino acids, in particular conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as argination, ubiquitination, or any other procedure such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine as well as D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by the indicated nucleic acid sequences.

The term "antigen binding unit" refers to immunoglobulin molecules (or "antibodies") and immunologically active portions of immune molecules, i.e., molecules that contain an antigen binding site that specifically binds to an antigen ("immunoreacts"). Also included within the term "antigen binding unit" are immunoglobulin molecules of various species origin, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (e.g., igG) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, which are interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, igG, igA, igM and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen binding function of antibodies can be performed by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". Also included within the term "antigen binding unit" are any polypeptide chain-containing molecular structure having a specific shape that conforms to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Examples of the antigen-binding unit include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, bivalent fragments (F (ab) 2 fragments) comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH and CH1 domains, an Fv fragment consisting of VL and VH domains of a single arm of an antibody; dAb fragments consisting of VH domains (Ward et al Nature,341:544-546,1989); and isolated Complementarity Determining Regions (CDRs) or any fusion proteins comprising such antigen binding units.

The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. The term "antibody" as used herein refers to an antigen binding protein of the immune system. The term "antibody" as referred to herein includes whole full length antibodies having an antigen binding region as well as any fragment thereof in which an "antigen binding portion" or "antigen binding region" remains, or a single chain thereof, e.g., a single chain variable fragment (scFv). "Natural antibody" refers to naturally occurring immunoglobulin molecules having a variety of structures, and refers to glycoproteins comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof, interconnected by disulfide bonds. The term "antibody" also includes all recombinant forms of antibodies (particularly the antibodies described herein), such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies as well as antibody fragments that bind to an antigen and derivatives described below. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The term "variable region or variable domain" refers to the domain of an antibody heavy or light chain that is involved in antigen binding of an antibody. VH and VL can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, typically arranged from amino-terminus to carboxyl-terminus in the following order: FR1-HCDR1 (LCDR 1) -FR2-HCDR2 (LCDR 2) -FR3-HCDR3 (LCDR 3) -FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated using a library of complementary VL or VH domains screened using VH or VL domains, respectively, from antibodies that bind the antigen. See, e.g., portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature352:624-628 (1991).

The term "hypervariable region" or "complementarity determining region" or "CDR" refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops") and/or contain residues that contact an antigen ("antigen contacts"). Typically, an antibody comprises six CDRs: three of VH (HCDR 1, HCDR2, HCDR 3) and three of VL (LCDR 1, LCDR2, LCDR 3).

Antibody fragments include, but are not limited to: (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab 'and Fab' -SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al 1989,Nature 341:544-546); (v) A F (ab') 2 fragment, a bivalent fragment comprising 2 linked Fab fragments; (vi) Single chain Fv molecule antigen binding sites (Bird et al 1988,Science 242:423-426; huston et al 1988, proc. Natl. Acad. Sci. U.S. A85:5879-5883); (vii) Bispecific single chain Fv dimers (PCT/US 92/09965); (viii) "diabodies" or "trisomy", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al 2000,Methods Enzymol.326:461-479; WO94/13804; holliger et al 1993,Proc.Natl.Acad.Sci.U.S.A 90:6444-6448); and (ix) scFv genetically fused to the same or different antibodies (Coloma & Morrison,1997,Nature Biotechnology 15,159-163).

"class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and some of these can be further divided into subclasses (allotypes), for example, igG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "Fc" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions.

Unless otherwise indicated herein, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al above.

The terms "fully anti," "full length antibody," "intact antibody" are used interchangeably to refer to a full length antibody having a structure substantially similar to the structure of a native antibody or having a heavy chain comprising an Fc region as defined herein or including an intact full length antibody having an antigen binding region. In particular embodiments, the application provides full length antibodies, the heavy and light chains of which may be full length (e.g., an antibody may comprise at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains) or may comprise an antigen binding portion (Fab, F (ab') 2, fv, or scFv). In other embodiments, the antibody heavy chain constant region is selected from, for example, igG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE. The choice of antibody type will depend on the immune effector function the antibody is designed to elicit. In constructing recombinant immunoglobulins, suitable amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for producing a wide variety of antibodies are known to those skilled in the art.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker),and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL. In some embodiments, flexible amino acids (G4S) are introduced against the VH and VL fragments of the antibody ₃ As a linker, a single chain variable fragment (scFv) is composed, the amino acid sequence of which confers specificity to the molecule for CS1 and forms the basis of the overall antigen binding unit of the application. Thus, the scFv can be used to design a range of different "antibody" molecules, including, for example, full length antibodies, fragments thereof such as Fab and F (ab') 2, scFv, fusion proteins (including scFv_Fc), multivalent antibodies, i.e., antibodies having more than one specificity for the same antigen or different antigens, e.g., bispecific T cell binding antibodies (BiTE), tri-antibodies, etc. (Cuesta et al, multivalent antibodies: when design surpasses evolution, trends in Biotechnology 28:355-362,2010). In some embodiments, the application includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having a human or murine immunoglobulin Fc region to produce bivalent proteins, thereby increasing the overall affinity and stability of the antibodies. In addition, the Fc portion allows direct conjugation of other molecules (including but not limited to fluorochromes, cytotoxins, radioisotopes, etc.) to antibodies, for example, for use in antigen quantification studies, in order to immobilize the antibodies for affinity measurement, for targeted delivery of therapeutic agents, testing Fc-mediated cytotoxicity using immune effector cells, and many other applications.

The term "single domain antibody (Single domain antibody, sdAb)" refers to a class of antibodies that lack the light chain and only the heavy chain variable region of the antibody, also known as nanobodies (nanobodies) due to their small molecular weight.

The term "single domain antibody" refers to a heavy chain variable domain comprising all or part of an antibody or all or part of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (domntis, inc., waltham, MA; see, e.g., U.S. patent No. 6248516).

The terms "monoclonal antibody", "mab" refer to an antibody obtained from a population of substantially homologous antibodies, i.e., individual antibodies comprising the population are identical and/or bind to identical epitopes, except for possible variant antibodies, e.g., comprising naturally occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants typically being present in minor amounts. In contrast to polyclonal antibody preparations (which typically include different antibodies directed against different determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, reference to "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring preparation of the antibody by any particular method. For example, it can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

For example, monoclonal antibodies of the application can be produced by a hybridoma method, which can be formed by isolating stimulated immune cells such as those from the spleen of an vaccinated animal. These cells (such as myeloma cells or transformed cells) can then be fused with immortalized cells, which are capable of unlimited replication in cell culture, thereby producing an immortalized, immunoglobulin-secreting cell line. The immortalized cell lines utilized are selected (whether they lack the enzymes necessary for the utilization of certain nutrients). Many such cell lines (such as myeloma) are known to those skilled in the art and include, for example: thymidine Kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPRT). These drawbacks allow: fusion cells are selected for their ability to grow on, for example, hypoxanthine aminopterin thymidine medium (HAT). Illustratively, the present application screens for 3 hybridoma antibodies 32a12, 37A3, 48G9 that bind hSLAMF 7.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain of the antibody is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. In certain embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In further embodiments, the chimeric antibody is a "type-switching" antibody, wherein the type or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, the chimeric antibody is a "humanized antibody".

The term "humanized" as used in connection with non-human antibodies, e.g., rodents or primates, is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof, that contains minimal sequences derived from a non-human immunoglobulin. "humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one (and typically two) variable domain, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, a "humanized antibody" may include mutations, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. Exemplary, the present application humanizes hybridoma antibodies 32a12, 37A3, 48G9 by CDR grafting to obtain hu32a12, hu37A3, hu48G9.

The term "parent antibody" or "parent immunoglobulin" includes unmodified antibodies which are then modified to produce variants. The parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody. A parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or an amino acid sequence encoding thereof. The term "parent antibody" or "parent immunoglobulin" as used herein includes murine or chimeric antibodies that have been modified to produce humanized antibodies.

The term "variant antibody" or "antibody variant" includes antibody sequences that differ from the parent antibody sequence by at least one amino acid modification compared to the parent. Variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to the parent antibody sequence. An antibody variant may refer to the antibody itself, a composition comprising said parent antibody, or an amino acid sequence encoding the same. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.

The term "amino acid modification" includes amino acid substitutions, additions and/or deletions, "amino acid substitutions" means the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. "amino acid insertion" means the addition of an amino acid at a particular position in the parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid at a particular position in a parent polypeptide sequence. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as: binding to the antigen.

The term "modification" refers to a change in the state or structure of a protein or polypeptide of the application. The manner of modification may be chemical, structural and functional.

The term "conservative modification" or "conservative sequence modification" means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody that contains the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications may be introduced into the antibodies of the application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions that replace an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids containing basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged acute side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions or in the framework regions of the antibodies of the application may be replaced with other amino acid residues of the same side chain family, and the altered antibodies (variant antibodies) may be tested for retained function.

All immunoglobulin heavy chain constant region positions discussed in this application are numbered according to the EU index of Kabat (Kabat et al, 1991,sequences of proteins of immunological interest, 5 th edition, united States Public Health Service, national Institutes of Health, bethesda, incorporated by reference in its entirety). The "EU index of Kabat" refers to the residue numbering of the human IgG1 EU antibody, as described by Edelman et al, 1969,Biochemistry 63:78-85.

The terms "anti-CS 1 antibody", "CS 1-recognizing antibody" refer to an antibody capable of binding CS1 with sufficient affinity, which can be used as a diagnostic and/or therapeutic agent for targeting CS1. In one embodiment, the anti-CS 1 antibody binds to less than about 10% of the non-CS 1 protein to which it is not associated, as determined by an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antigen binding units of the application that target CS1 bind to an Ig-like V-type domain distal to the extracellular domain of hCS1, or a membrane-proximal Ig-like C2-type domain of the extracellular domain.

In the present application, it is described that hybridoma antibodies are obtained by immunizing mice with the immunogen hSLAMF7-avi-His recombinant protein using techniques conventional in the art for hybridoma antibody preparation. Humanized modification is also carried out on the hybridoma antibody by a CDR grafting method to obtain the humanized antibody. These molecules exhibit specificity. In specific embodiments, the antibody recognizes CS1 protein. Illustratively, the CS1 antibody recognizes CS 1-expressing cells, e.g., tumor cells MM.1S cells, RPMI 8226 cells, NCI-H929 cells, NK cells. In specific embodiments, the CS1 antibody does not recognize CS1 negative cells, e.g., HEK293, WI38 cells. In the present application, CS1 herein refers to CS1 of a human, CS1 of a mouse or CS1 of a monkey unless otherwise specified.

The results presented herein highlight the specificity, sensitivity and utility of the antibodies of the application in targeting CS 1.

The present application provides an antigen binding unit that recognizes CS1 comprising an amino acid sequence comprising SEQ ID NO:3, 13, 23, and/or a heavy chain CDR1 comprising any one of the amino acid sequences of SEQ ID NO:4, 14, 24, 32, and/or a heavy chain CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 15, 25, 41. In another aspect, the application provides an antigen binding unit that binds CS1, or a fragment thereof, comprising an amino acid sequence comprising SEQ ID NO:8, 18, 28, and/or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, 19, 29, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10 Light chain CDR3 of any one of amino acid sequences 20, 30. In another aspect, the application provides an antigen binding unit that binds CS1, or a fragment thereof, comprising an amino acid sequence comprising SEQ ID NO:3, 13, 23, and/or a heavy chain CDR1 comprising any one of the amino acid sequences of SEQ ID NO:4, 14, 24, 32, and/or a heavy chain CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 15, 25, 41, and/or a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 18, 28, and/or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, 19, 29, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10 Light chain CDR3 of the amino acid sequence of any one of 20, 30. Preferably, the CS 1-binding antibody or fragment thereof comprises a polypeptide comprising SEQ ID NO:3, 13, 23, and a heavy chain CDR1 comprising any one of the amino acid sequences of SEQ ID NO:4, 14, 24, 32, and a heavy chain CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 5, 15, 25, 41, and/or a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 18, 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, 19, 29, and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10 Light chain CDR3 of the amino acid sequence of any one of 20, 30. More preferably, the antibody or fragment thereof that binds CS1 comprises an amino acid sequence comprising SEQ ID NO:3, 13, 23, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, 14, 24, 32, and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, 15, 25, 41, and a heavy chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 18, 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, 19, 29, and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10 Light chain CDR3 of the amino acid sequence of any one of 20, 30.

In another aspect, the application provides an antigen binding unit that recognizes CS1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 36 or 40.

In another aspect, the application provides an antigen binding unit that recognizes CS1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26, 34, 38 or 43.

Considering that each of these heavy and light chain variable region sequences can recognize CS1, the heavy and light chain variable region sequences can be "mixed and matched" to produce an anti-CS 1 binding molecule of the application.

In another aspect, the application provides an antigen binding unit that recognizes CS1 comprising a polypeptide selected from the group consisting of a heavy chain variable region having SEQ ID NO:1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:6, an amino acid sequence shown in figure 6; the heavy chain variable region has the amino acid sequence of SEQ ID NO:11 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no; the heavy chain variable region has the amino acid sequence of SEQ ID NO:21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no; the heavy chain variable region has the amino acid sequence of SEQ ID NO:31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no; the heavy chain variable region has the amino acid sequence of SEQ ID NO:36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:38, and a nucleotide sequence shown in seq id no; the heavy chain variable region has the amino acid sequence of SEQ ID NO:40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 43.

In another aspect, the application provides variants that recognize the antigen binding unit of CS 1. The application thus provides an antigen binding unit having a heavy and/or light chain variable region that is at least 80% identical to the variable region sequence of a heavy or light chain. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including for example 80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% and 100%. The variant can be obtained by taking the antibody as a female parent antibody through methods such as yeast library screening, phage library screening, point mutation and the like.

In another aspect, the application provides an antigen binding unit that recognizes the same epitope as the aforementioned antigen binding unit against CS 1; or binding to the Ig-like V-type domain of CS 1; or Ig-like C2-type domain binding CS 1. The antigen binding units for recognizing CS1 can specifically bind CS1 positive cells and not CS1 negative cells, and show good therapeutic potential. The scFv of the antigen binding unit of the partial recognition CS1 is stable in structure and is not easy to aggregate.

In another aspect, the application provides an antigen binding unit that recognizes CS1, which is a hybridoma, humanized, chimeric, or fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is a full anti, scFv, single domain antibody, fv fragment, fab 'fragment, (Fab') 2 fragment, dAb fragment or multifunctional antibody.

The anti-CS 1 antibodies provided herein may be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art. Including, for example, ELISA, biacore, microplate reader and flow cytometer analyses. Suitable assays are described in detail in the examples.

The term "antigen" refers to a substance that is recognized and specifically bound by an antigen binding unit. Antigens may include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. An "antigen" may also refer to a molecule that initiates an immune response. Such an immune response may involve antibody production or activation of specific immunocompetent cells (or both). Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, may be used as an antigen. Antigens useful in the present application include human, mouse, monkey SLAMF7 recombinant proteins.

The term "affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an inherent binding affinity that reflects a reaction between members of a binding pair (e.g., antibodies and antigens) of 1:1 interact. The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the determination of affinity of antibodies using Biacore as described herein. The "affinity" of an antibody herein for CS1 is expressed as the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody to its antigen, the weaker its binding affinity for that particular antigen. Illustratively, the present application measures the affinity of CS1 antibodies to SLAMF7 recombinant proteins of different species (exemplified, human, mouse, monkey) with KD values ranging from 940pM to 107nM.

The term "EC50" refers to the concentration that gives 50% of the maximum effect concentration (concentration for 50%of maximal effect,EC50). The application detects the EC50 value of CS1 antibody and recombinant protein hSLAMF7-avi-His by ELISA method, which is in the range of 0.01-0.058 mug/ml; EC50 values of CS1 antibody and CS1 positive cells MM.1S cells were measured with a flow cytometer and ranged from 0.05-1.76. Mu.g/ml.

In some embodiments, the application also detects binding of CS1 antibodies to CS1 of different species (exemplified, human, mouse, monkey) using a microplate reader, both antibodies bind to human CS1, not to mouse CS1, and antibodies 37A3 and 48G9 also bind to monkey CS1.

The term "epitope" is also known as an "epitope," or "epitope" or "antigenic determinant," and includes any determinant or region capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, including specific amino acids that are in direct contact with the antibody. For example, an epitope may consist of a contiguous sequence of the CS1 protein sequence, or may consist of a discontinuous three-dimensional structure of the CS1 protein sequence. Illustratively, the antigen used herein is CS1 in a human, murine, or monkey. According to the application, the binding epitope of the CS1 antibody and the human CS1 is analyzed to obtain the Ig-like V-type structural domain of the binding epitope of the human CS1, and the far membrane end; or at the Ig-like C2-type domain, proximal to the membrane, of human CS1.

The application also provides immunoconjugates comprising the antibodies described herein, and functional molecules linked thereto. The antibody and the functional molecule can form a conjugate through covalent connection, coupling, adhesion, crosslinking and the like.

The functional molecule is selected from: molecules targeting tumor surface markers, molecules inhibiting tumors, molecules targeting surface markers of immune cells or detectable markers. In some embodiments, the molecule that targets a surface marker of an immune cell is an antigen binding unit (e.g., an antibody) that binds to a surface marker of a T cell, which forms a bifunctional antigen binding unit (e.g., a bifunctional antibody) with which the T cell participates.

The terms "connected" or "fused" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components by any means including chemical conjugation or recombinant means. "in-frame fusion" refers to joining two or more Open Reading Frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct reading frame of the original ORF. Thus, the resulting recombinant fusion protein is a single protein containing two or more fragments corresponding to the polypeptide encoded by the original ORF (these fragments are not typically so linked in nature). Although the reading frames are thus contiguous throughout the fusion fragment, the fragments may be physically or spatially separated by, for example, in-frame joining sequences (e.g., "flexon").

In another aspect, the application provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate of the application. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

The application also provides nucleic acid molecules encoding the aforementioned antibodies, preferably the nucleic acid molecules of the application are selected from SEQ ID NO. 2, 12, 22, 33, 37 or 42 encoding the heavy chain variable region and/or from SEQ ID NO. 7, 17, 27, 35, 39 or 44 encoding the light chain variable region. More preferably, it is a nucleic acid molecule comprising the heavy chain variable region sequence of SEQ ID NO. 2 and comprising the light chain variable region sequence of SEQ ID NO. 7; or a heavy chain variable region sequence comprising SEQ ID NO. 12 and a light chain variable region sequence comprising SEQ ID NO. 17; or a heavy chain variable region sequence comprising SEQ ID NO. 22 and a light chain variable region sequence comprising SEQ ID NO. 27; or a heavy chain variable region sequence comprising SEQ ID NO. 33 and a light chain variable region sequence comprising SEQ ID NO. 35; or a heavy chain variable region sequence comprising SEQ ID NO. 37, and a light chain variable region sequence comprising SEQ ID NO. 39; or a heavy chain variable region sequence comprising SEQ ID NO. 42 and a light chain variable region sequence comprising SEQ ID NO. 44.

In one embodiment, one or more vectors (e.g., expression vectors) comprising the above nucleic acids are provided.

The application also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

The term "cell" refers to a cell of human or non-human origin that is animal. In one embodiment, the engineered cell or engineered cell refers to a T cell that expresses a CS 1-CAR.

The term "host" or "subject" refers to a recipient that receives a graft transplant, and in some embodiments, may be an individual, such as a human, that receives an exogenous cell implant. In some embodiments, the "subject" may be a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. The subject may be suspected of having a disease characterized by cellular proliferation or having a disease characterized by cellular proliferation, diagnosed as having a disease characterized by cellular proliferation, or a control subject that is confirmed to not have a disease characterized by cellular proliferation. In some embodiments, the subject is suffering from or likely to suffer from an immune disorder, such as an autoimmune disorder, or is treated with a transplant. The term "patient" is a subject suffering from or at risk of suffering from a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.

The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include transformed primary cells and their progeny (regardless of the number of passages). The nucleic acid content of the offspring may not be exactly the same as the parent cell and may contain mutations. Included herein are mutant progeny that have the same functional or biological activity as screened or selected for in the originally transformed cell.

In one embodiment, a method of making an anti-CS 1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).

In one embodiment, a host cell comprising a nucleic acid encoding an antibody as described above is provided. The host cell comprises (e.g., is transduced with): (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is a eukaryotic cell, e.g., 293T cell.

In another embodiment, the host cell may also express a chemokine receptor.

In another embodiment, the host cell may also express a safety switch.

In a preferred embodiment, the host cell is administered in combination with an agent that enhances its function, preferably in combination with a chemotherapeutic agent; and/or the host cell is administered in combination with an agent that ameliorates one or more side effects associated therewith; and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor that targets a site other than CS 1.

In some embodiments, the host cell is an immune effector cell.

The term "immune effector cells" refers to cells involved in an immune response, such as T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, and the like, that produce an immune effect. In some embodiments, the immune effector cells are T cells, NK cells, NKT cells. In some embodiments, the T cells may be autologous T cells, xenogeneic T cells, allogeneic T cells. In some embodiments, the NK cells can be autologous NK cells or allogeneic NK cells. The term "CIK cell", i.e. Cytokine-Induced Killer (CIK), is a novel immunocompetent cell, and has strong CIK proliferation capacity, strong cytotoxic effect and certain immune characteristics. Since the cell expresses two membrane protein molecules of CD3 and CD56 simultaneously, the cell is also called NK cell (natural killer cell) like T lymphocyte, and has the advantages of strong anti-tumor activity of the T lymphocyte and non-MHC restriction tumor killing of the NK cell.

The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that have no immune effect that have acquired immune effector cell function after being artificially modified or stimulated with a stimulus. For example, 293T cells are artificially modified to have the function of immune effector cells; such as stem cells, are induced in vitro to differentiate into immune effector cells.

The T cells described herein may be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue and tissue from infection sites, ascites, pleural effusions, spleen tissue and tumors, may also be populations of cells having particular phenotypic characteristics obtained by sorting or the like, or mixed populations of cells having different phenotypic characteristics, such as "T cells" may be cells comprising at least one subpopulation of T cells: memory stem cell-like T cells (stem cell-like memory T cells, tscm cells), central memory T cells (Tcm), effector T cells (Tef, teff), regulatory T cells (tregs), and/or effector memory T cells (Tem). In some cases, a "T cell" may be a T cell of a particular subtype, such as an αβ T cell, γδ T cell. In some cases, T cells may be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as ficoll (tm) isolation. T cells may be any type of T cell, and may be at any stage of development, including but not limited to cd4+/cd8+ double positive T cells, cd4+ helper T cells, such as Th1 and Th2 cells, cd8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, astronomical T cells, and the like. The T cells may be cd8+ T cells or cd4+ T cells. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells collected by apheresis may be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps. In one embodiment, the derived cells may be derived from a healthy donor, or from an individual diagnosed with cancer.

The terms "activate" and "activating" are used interchangeably and may refer to the process by which a cell transitions from a resting state to an active state. The process may include a response to a phenotypic or genetic change in the antigen, migration, and/or functionally active state. For example, the term "activation" may refer to the process of stepwise activation of T cells. The activation process is co-regulated by the first stimulus signal and the co-stimulus signal. "T cell activation" or "T cell activation" refers to the state of a T cell that is stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function. Using CD3/CD28 magnetic beads, either in vitro or in vivo antigen stimulation will have an effect on the degree and duration of T cell activation. In one embodiment, the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.

The term "peripheral blood mononuclear cells" (peripheral blood mononuclear cell, PBMC) refers to cells having a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.

The term "pluripotent stem cell" has the potential to differentiate into any of three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, genitourinary tissue, etc.) or ectoderm (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also encompasses "induced pluripotent stem cell" or "iPSC", which is a type of pluripotent stem cell derived from a non-pluripotent cell. In one embodiment, the pluripotent stem cells are derived from cells transformed by reprogramming somatic cells to have pluripotent stem cell characteristics. Such "iPS" or "iPSC" cells may be produced by inducing the expression of certain regulatory genes or by exogenously applying certain proteins.

"pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. For example, human pluripotent stem cells express at least several of the following markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, sox2, E-cadherin, UTF-1, oct4, rex1 and Nanog. Having a cell morphology associated with pluripotent stem cells is also a feature of pluripotent stem cells.

The term "engineering" refers to a comprehensive scientific technique that applies principles and methods of cell biology and molecular biology to alter, by some engineering means, genetic material within a cell or to obtain a cellular product at the cellular global or organelle level, as desired. In one embodiment, the engineering refers to one or more alterations of a nucleic acid, such as a nucleic acid within the genome of an organism. In one embodiment, the engineering refers to alterations, additions and/or deletions of genes. In one embodiment, the engineered cell or the engineered cell may also refer to a cell having added, deleted, and/or altered genes.

The terms "genetic modification", "genetically engineered" or "modified" refer to a method of modifying a cell, including but not limited to, the modification of a gene in a coding or non-coding region or an expression regulatory region thereof by means of gene editing; or by endonuclease and/or antisense RNA techniques; or increasing the introduction of exogenous proteins and/or complexes, small molecule inhibitors to alter the protein expression levels of the gene to create a gene defect. In some embodiments, the modified cells are stem cells (e.g., hematopoietic Stem Cells (HSCs) or progenitor cells, embryonic stem cells (ESs), induced Pluripotent Stem (iPS) cells), lymphocytes (e.g., T cells), which may be obtained from a subject or donor. The cells may be modified to express an exogenous construct, such as a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR), which may be integrated into the cell genome.

By "TCR silencing" is meant that the endogenous TCR is not expressed or is underexpressed.

By "MHC silencing" is meant that endogenous MHC is not expressed or is underexpressed.

By "low expression" is herein meant that the level of protein and/or RNA expressed by the target gene in the engineered cell is lower than the level of expression prior to the cell engineering process. In particular embodiments, low expression of B2M or TCR or CS1 refers to a decrease in expression of B2M or TCR or CS1 in a cell of at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of the protein in the cells may be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting (Western Blotting) or flow cytometry using antibodies specific for B2M or TCR or CS 1.

The term "B2M" is a beta-2 microglobulin, also known as B2M, which is the light chain of an MHC class I molecule. Plays an important role in the transplantation reaction, and rejection is mediated by T cells that react to histocompatibility antigens on the surface of the implanted tissue.

The term "T Cell Receptor (TCR)" mediates T cell recognition of specific Major Histocompatibility Complex (MHC) -restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. TCRs fall into two categories: TCR1 and TCR2; TCR1 consists of two chains, γ and δ, and TCR2 consists of two chains, α and β. The term "TRAC" refers to the constant region of the TCR alpha chain.

The term "gene editing" refers to genetic engineering techniques that use site-specific nucleases to insert, knock out, modify or replace DNA at specific locations in the genome of an organism, altering the DNA sequence. This technique is sometimes referred to as "gene clipping" or "genome engineering". Gene editing can be used to achieve accurate, efficient gene knockouts or gene knockins.

Nuclease-directed genomic targeted modification techniques typically consist of a DNA recognition domain and a non-specific endonuclease domain, with the recognition of the target site by the DNA recognition domain, localization of the nuclease to the genomic region to be edited, and subsequent cleavage of the DNA duplex by the non-specific endonuclease, causing DNA fragmentation self-repair mechanisms, thereby initiating mutation of the gene sequence and promoting homologous recombination. The endonuclease may be a Meganuclease (Meganuclease), a zinc finger nuclease, a CRISPR/Cas9 nuclease, an MBBBD-nuclease, or a TALEN-nuclease. In preferred embodiments, the endonuclease is a CRISPR/Cas9 nuclease, TALEN-nuclease. Gene knockout techniques using nucleases include CRISPR/Cas9 techniques, ZFN techniques, TALE techniques, and TALE-CRISPR/Cas9 techniques, base Editor techniques, guided editing techniques, and/or homing endonuclease techniques.

One embodiment of the application employs CRISPR/Cas9 technology to prepare UCAR-T cells. The term "CRISPR (Clustered regularly interspaced short palindromicrepeats)" refers to short palindromic repeats at regular clustered intervals. The term "Cas9 (CRISPRassociated nuclease)" is a CRISPR-associated nuclease, a RNA-guided technique for editing a targeted gene with a Cas9 nuclease. In the context of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. After CRISPR complex is formed, specific loci of a genome can be cut under the action of cas9 enzyme, and gene mutation is introduced; expression of the gene may also be regulated, such as by activation or inhibition. A target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In general, a guide sequence (gRNA) is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct the sequence-specific binding of a CRISPR complex to the target sequence. The application relates to a sequence of gRNA, which can be a targeted DNA sequence or a complete Cas9 guide sequence formed by ribonucleotides corresponding to the DNA and crRNA, tracrRNA. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. The term "sgRNA" refers to short gRNA.

CRISPR/Cas transgenes can be delivered by vectors (e.g., AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electrotransport.

The application obtains the universal T cells or the universal CAR-T cells by knocking out genes TRAC and B2M. In one embodiment, the exons of the corresponding coding genes in the constant region of one or both of the alpha and beta strands of B2M, TCR are knocked out using the CRISPER/Cas technique, respectively. In one embodiment, the gRNA used to knock out the TCR is selected from the sequences set forth in SEQ ID NOs 76, 77, 78, 79, 80, 81, 82 and/or 83. In one embodiment, the engineered T cell B2M gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the sequences set forth in SEQ ID NOs 84, 85, 86 and/or 87.

In one embodiment, the cell CS1 gene is knocked out, reducing killing of CS1-CAR-T cells on themselves. Illustratively, the CS1 gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the sequences set forth in SEQ ID NOs 88, 89, 90, 91, 92, 93, 94 and/or 95.

By "inhibiting" or "suppressing" expression of B2M or TCR or CS1 is meant reducing expression of B2M or TCR or CS1 in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of the protein in the cells may be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting (Western Blotting) or flow cytometry using antibodies specific for B2M or TCR or CS 1.

In one embodiment of the application, specific CAR-T cells are constructed first, and then the CRISPER/Cas9 technology is utilized to knock out the endogenous TRAC, B2M and/or CS1 of the CAR-T cells to construct corresponding UCAR-T. In one embodiment, the CRISPER/Cas9 technique is used to knock out endogenous TRAC, B2M, and/or CS1 to construct universal T cells, and then to express specific CARs to construct UCAR-T cells. In one embodiment, the CRISPER/Cas9 technique knocks out endogenous TRAC, B2M and/or CS1 and expression of a specific CAR are operated simultaneously to construct UCAR-T cells.

The term "transfection" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and their progeny. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (biolistics).

The terms "nucleic acid molecule encoding", "encoding DNA sequence" and "encoding DNA" refer to the sequence or order of deoxyribonucleotides along the strand of a deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain.

The term "sequence" as used herein when used in reference to a nucleotide sequence or polynucleotide sequence may include DNA or RNA, and may be single-stranded or double-stranded.

The term sequence "identity" as used herein determines the percent identity by comparing two optimally matched sequences over a comparison window (e.g., at least 20 positions), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps), such as 20% or less (e.g., 5 to 15%, or 10 to 12%) of the gap(s) for the optimally matched two sequences compared to the reference sequence (which does not comprise additions or deletions). The percentage is typically calculated by determining the number of positions at which the same nucleobase or amino acid residue occurs in both sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to produce the percentage of sequence identity.

The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as plasmids, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). For expression of the protein, the nucleic acid encoding the antigen binding unit of the application may be integrated into an expression vector. Expression vectors for use in the present application include, but are not limited to, expression vectors that enable proteins in mammalian cells, bacteria, insect cells, yeast and in vitro systems.

The term "vector" as used herein is a composition comprising an isolated nucleic acid and useful for delivering the isolated nucleic acid into the interior of a cell. Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds, such as polylysine compounds, liposomes, and the like, that facilitate transfer of nucleic acids into cells may also be included.

The term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously by the organism itself, or that is not expressed at levels sufficient to achieve overexpression.

The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., derived from the organism itself.

The term "chimeric receptor", i.e., a fusion molecule comprising a DNA fragment of different origin or a cDNA or polypeptide fragment corresponding to a protein, linked by genetic recombination techniques, includes an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to: chimeric Antigen Receptor (CAR), chimeric T Cell Receptor (TCR), T cell antigen coupler (TAC).

The term "chimeric T cell receptor" consists of a TCR subunit associated with an antigen binding domain (e.g., an antibody domain), wherein the TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane domain, a stimulatory domain of a TCR intracellular signaling domain; the TCR subunit and the antibody domain are operably linked. In a specific embodiment, the antigen binding domain of the chimeric T cell receptor comprises a CS1 antigen recognition unit according to the application.

The term "T cell antigen coupler (T cell antigen coupler, TAC)", includes three functional domains: (1) An antigen binding domain comprising a single chain antibody, a designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting group; (2) An extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; (3) a transmembrane region and an intracellular region of a CD4 co-receptor. In a specific embodiment, the TAC antigen-binding domain comprises a CS1 antigen-recognition unit according to the application.

The application provides a T cell capable of obviously killing target cells. In particular embodiments, the application provides CAR-T cells, e.g., hu32a12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells, comprising the CS1 antigen recognition unit. The CS1-CAR-T cell has any one of the following advantages:

(1) In vitro and in vivo experiments, after co-incubation of the CS1-CAR-T cells with CS 1-positive target cells, the CS1-CAR-T cells produce significant specific proliferation.

(2) The CS1-CAR-T cells can specifically kill CS 1-positive target cells. Illustratively, the CS1-CAR-T cells are capable of specifically killing CS 1-positive tumor cells. In particular embodiments, the CS1-CAR-T cells specifically kill CS 1-positive multiple myeloma in vitro and in vivo experiments. Since CS1 is a member of the lymphocyte activating molecule family, involved in NK cell activating function, CS1 is expressed on NK cells; illustratively, in vitro and in vivo experiments, the CS1-CAR-T cells specifically kill host NK cells, or the CS1-CAR-T cells are resistant to killing of autologous or allogeneic immune cells (e.g., T cells, CAR-T cells) by the host NK cells, increase the persistence and/or the survival rate of the autologous or allogeneic immune cells in the presence of the host immune cells. Illustratively, in vitro and in vivo experiments, soluble CS1 does not affect the killing effect of the CS1-CAR-T cells on CS 1-positive target cells.

(2) Cytokine secretion: in vitro and in vivo experiments, the CS1-CAR-T cells can be obviously activated after being incubated with CS1 positive target cells so as to secrete cytokines TNF-alpha, IL-2 or IFN-gamma; in vitro and in vivo experiments, the CS1-CAR-T cells did not produce significant secretion of the non-specific cytokines TNF- α, IL-2 or IFN- γ upon co-incubation with CS1 negative cells; in vitro and in vivo experiments, the level of cytokine IL-6 secretion was low following co-incubation of the CS1-CAR-T cells with monocytes and CS1 positive target cells.

(3) And (3) detecting the exhaustion degree: in vitro and in vivo experiments, after co-incubation of the CS1-CAR-T cells with CS 1-positive target cells, the CS1-CAR-T cell depletion markers PD-1, TIM-3, LAG-3 are expressed less; the CS1-CAR-T cells have a lower degree of CD 3-zeta phosphorylation during in vitro culture without target cell stimulation.

In particular embodiments, the application provides universal T cells (CS 1-UCAR-T) comprising the CS1-CAR with endogenous TCR and B2M double knockouts, and endogenous CS1-UCAR-T cells with CS1, TCR and B2M triple knockouts. The double-knockout or triple-knockout CS1-UCAR-T cells have any of the following advantages: specifically killing CS1 positive target cells. Illustratively, the CS1-UCAR-T cells are capable of specifically killing CS 1-positive tumor cells. In specific embodiments, the CS1-UCAR-T cells specifically kill CS 1-positive multiple myeloma cells in vitro and in vivo experiments. Illustratively, in vitro and in vivo experiments, the CS1-UCAR-T cells specifically kill host NK cells, or the CS1-UCAR-T cells are resistant to killing of autologous or allogeneic immune cells (e.g., T cells, CAR-T cells, CS 1-UCAR-T) by the host NK cells, increase the persistence and/or the engraftment rate of the autologous or allogeneic immune cells in the presence of the host immune cells.

The term "chimeric antigen receptor" (CAR) includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain comprises an antigen-binding unit of the application that recognizes CS1, or comprises a Fv, fab, fab ', fab ' -SH, F (ab ') 2, scFv, or multispecific antibody derived from an antigen-binding unit of the application that recognizes CS 1.

Intracellular signaling domains include functional signaling domains of stimulatory molecules and/or co-stimulatory molecules; or all intracellular portions comprising stimulatory molecules and/or co-stimulatory molecules, or all native intracellular signaling domains, or functional fragments or derivatives thereof. In one aspect, the stimulatory molecule comprises a cd3ζ chain associated with the T cell receptor complex; in one aspect, the intracellular signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD 137), CD27, and/or CD28. In certain embodiments, the sets of polypeptides are linked to each other. Illustratively, the CS 1-targeted CAR comprises SEQ ID NO: 50. 51 or 52, and exemplary CS 1-targeting CARs comprise the antigen binding domain shown in SEQ ID NO: 45. 46 or 47. The above SEQ ID NO: 45. 46, 47 and intracellular domains a person skilled in the art may select conventional transmembrane and intracellular domains for substitution and fall within the scope of the application.

The functional signaling domain of a stimulatory molecule, also known by the term "primary signaling domain", modulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signal domain is triggered by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain acting in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or a signaling motif of ITAM. Examples of primary signal domains comprising ITAM that are particularly useful in the present application include, but are not limited to, sequences derived from tcrζ, cd3ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66 d. In a particular example of a CAR of the application, the intracellular signaling domain in any one or more of the CARs of the application comprises an intracellular signaling sequence, such as the primary signaling domain of CD3 ζ.

The term "costimulatory signal domain" refers to a "costimulatory molecule" which refers to a signal that, in combination with a cell-stimulating signal molecule, such as TCR/CD3, results in the up-or down-regulation of T cell proliferation and/or a key molecule. Is a cognate binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cells, including but not limited to cell proliferation. Costimulatory molecules are cell surface molecules or ligands thereof that are non-antigen receptors required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18) and 4-1BB (CD 137).

Illustratively, the signaling domain of a CS 1-targeted CAR includes cd3ζ. Exemplary, signaling domains of CS 1-targeted CARs include the CD28 intracellular domain, CD137 intracellular domain. In a specific embodiment, the CD28 intracellular domain comprises the sequence shown as SEQ ID NO. 73 and the CD137 intracellular domain comprises the sequence shown as SEQ ID NO. 55. In the present application, "CD3 ζ" is used interchangeably with "CD3Z" and "CD 3Z". In a specific embodiment, CD3 zeta is a human CD3 zeta molecule comprising the sequence shown in SEQ ID NO: 56.

In particular embodiments, the CAR comprises an optional leader sequence. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to a cell membrane. In one embodiment, the leader sequence comprises the sequence shown as SEQ ID NO. 71.

In some embodiments, the extracellular antigen-binding region of the CAR comprises the antigen-binding unit and a linker fragment (also referred to as a hinge, spacer, or linker) of the application that recognizes CS 1. The linker fragment may be considered to be part of a CAR for providing flexibility to the extracellular antigen-binding region. The connecting segments may be of any length. Illustratively, in one embodiment, the CAR comprises a hinge domain that is a CD8 a hinge, preferably the CD8 a hinge domain comprises the amino acid sequence shown in SEQ ID No. 53.

The Transmembrane (TM) domain of the CAR (or referred to as a structural region) may anchor the CAR to the plasma membrane of the cell. Illustratively, TM is a CD8 or CD28 transmembrane domain. In a preferred embodiment, the TM is a human CD8 transmembrane domain or a human CD28 transmembrane domain. Preferably, the CD8 transmembrane domain comprises the amino acid of SEQ ID NO. 54 and the CD28 transmembrane domain comprises the amino acid of SEQ ID NO. 72.

The term "interferon" is a cytokine produced by the immune system, and is mainly classified into three types of alpha, beta and gamma, and has antiviral, antitumor and immunoregulatory effects.

The term "interleukin-2 (interleukin 2, IL-2)" is a cytokine of the chemokine family, and has an important role in the immune response of the body, antiviral infection, etc.

The term "interleukin 6 (interleukin 6, IL-6)" is a widely functional pleiotropic cytokine. IL-6 is a biomarker for cytokine storms.

The term "T cell depletion" is a state of T cell dysfunction that manifests itself as progressive loss of function, changes in gene expression profile, and sustained secretion of inhibitory cytokines. Biomarkers of T cell depletion include PD-1, TIM-3, LAG-3, and the like.

The antibodies, the immunological conjugates comprising the antibodies, the chimeric receptors and the host cells can be used for preparing pharmaceutical compositions or diagnostic reagents. The composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of the antibody, immunological conjugate, chimeric receptor, nucleic acid or host cell. The term "pharmaceutically acceptable" means that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. The pharmaceutically acceptable carrier may be one of the carriers conventionally used and is limited only by chemical and physical considerations, such as solubility and non-reactivity with the active agent, as well as the route of administration. Pharmaceutically acceptable carriers described herein, such as adjuvants, adjuvants and diluents, are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is a carrier which is harmless under the conditions of use and has no toxic or side effects. The pharmaceutical compositions of the present application are in a variety of suitable dosage forms. Methods of preparing administrable (e.g., parenteral) compositions are known or apparent to those skilled in the art.

In some embodiments, the composition comprises a chemotherapeutic agent.

The composition of the present application may be formulated into various dosage forms as required, and the dosage beneficial to the patient may be determined by the physician according to the type, age, weight and general condition of the patient, the mode of administration, etc. The mode of administration may be, for example, parenteral (e.g., injection) or other therapeutic.

"parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection or infusion techniques.

The engineered cells of the application can be administered to a subject in any suitable manner. Preferably, the antibodies of the application, immunogenic conjugates comprising the antibodies, chimeric receptors, host cells are administered by injection (e.g., subcutaneously, intravenously, intratumorally, intraarterially, intramuscularly, intradermally, intraperitoneally or intrathecally). Preferably, the antibodies, immunoconjugates comprising the antibodies, chimeric receptors, host cells of the application are administered intravenously. Suitable pharmaceutically acceptable carriers for injectable antibodies, immunological conjugates comprising the antibodies, chimeric receptors, host cells of the application may include any isotonic carrier, for example, physiological saline (about 0.90% w/v NaCl in water, about 300mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), ambient temperature, or an electrolyte solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

An "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to prevent or treat a disease (cancer) in an individual. The effective dose for therapeutic or prophylactic use will depend on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the discretion of the prescribing physician. The size of the dosage will also depend on the active substance selected, the method of administration, the time and frequency of administration, the presence, nature and extent of adverse side effects that may accompany the administration of a particular active substance, and the desired physiological effect. One or more rounds, or multiple administrations of the antibodies of the application, immunological conjugates comprising the antibodies, chimeric receptors, host cells may be required as determined by the prescribing physician or skilled artisan.

Embodiments of the application also include removing lymphocytes from a mammal prior to administration of an antibody, an immunological conjugate comprising the antibody, a chimeric receptor, a host cell of the application, including but not limited to non-myeloablative lymphocyte depletion chemotherapy, systemic irradiation, and the like.

The term "treatment" refers to interventions that attempt to alter the course of a disease, both prophylactic and clinical, as well. Therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of progression of a disease, improving or alleviating a condition, alleviating or improving prognosis, and the like. In specific embodiments, the engineered T-cells provided herein are capable of inhibiting tumor cell proliferation, and/or inhibiting tumor cell proliferation in vivo, tumor volume increase.

The term "prevention" refers to intervention prior to attempting to develop a disease such as rejection by cell transplantation.

The application provides an antibody, CAR, nucleic acid, recombinant expression vector, host cell, cell collection or pharmaceutical composition of the application for use in the treatment or prevention of a mammalian tumor.

The provided engineered T cells of the application are useful for the treatment, prevention or amelioration of autoimmune diseases or inflammatory diseases, particularly inflammatory diseases associated with autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis (arthritis chronica progrediente) and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spinal arthropathy (including ankylosing spondylitis), reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, start-stop point inflammation, hypersensitivity reactions (including airway hypersensitivity and skin hypersensitivity reactions) and allergies. The engineered T cells provided herein are useful for the treatment and prevention of conditions including autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure erythrocyte anemia, and idiopathic thrombocytopenia), systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory muscle diseases (dermatomyositis), periodontitis, polychondritis, scleroderma, wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, stefan Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel diseases (including, for example, ulcerative colitis, crohn's disease, and irritable bowel syndrome), endocrine ocular diseases, graves ' disease, sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes (type I diabetes), uveitis, keratoconjunctivitis sicca, and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, peri-prosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome), for example including idiopathic nephrotic syndrome or minuscular nephropathy), multiple myeloma, other types of tumors, inflammatory diseases of the skin and cornea, myositis, loosening of bone implants, metabolic disorders such as obesity, atherosclerosis and other cardiovascular diseases including dilated cardiomyopathy, myocarditis, type II diabetes and dyslipidemia, and autoimmune thyroid diseases including hashimoto's thyroiditis, medium and small vascular primary vasculitis, macrovasculitis including giant cell arteritis, suppurative sweat gland, neuromyelitis optica, sjogren's syndrome, behcet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathy, immune kidney, liver and thyroid diseases, inflammatory and atherosclerosis, auto-inflammatory fever syndrome, immune hematologic disorders, and bullous diseases of the skin and mucous membranes.

The engineered T-cells provided herein can be used to treat, prevent or ameliorate asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.

The engineered T cells of the application may be administered as the sole active ingredient or in combination (e.g., as an adjuvant or in combination) with other drugs such as immunosuppressants or immunomodulators or other anti-inflammatory or other cytotoxic or anticancer agents, e.g., to treat or prevent diseases associated with immune disorders.

The tumor of the present application may be any tumor including acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal or middle ear cancer, oral cancer, vulvar cancer, chronic Lymphocytic Leukemia (CLL), chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (such as head and neck squamous cell carcinoma), hodgkin lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (such as non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin lymphoma, B-chronic lymphocytic leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), pre-B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell lymphoma, acute Lymphoblastic Leukemia (ALL), burkitt lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer. Preferably, the tumor is characterized by CS1 expression, and more preferably is multiple myeloma characterized by CS1 expression.

"tumor antigen" refers to an antigen that is either newly emerged or overexpressed during the development, progression, or development of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present application is cancer.

The application provides a method for expressing CS1-CAR by genetic engineering of T cells for obviously killing target cells; a method for preparing the genetically engineered T cells is provided. In some embodiments, the engineered T cell is genetically engineered to express a CS1-CAR. The application also provides a T cell which not only can obviously kill tumor cells, but also can resist killing of autologous or allogeneic NK cells. In some embodiments, the engineered T cells are genetically engineered to express a CS1-CAR, and endogenous CS1 is knocked out using gene editing techniques. In some embodiments, the engineered T cells are genetically engineered to express a CS1-CAR, and endogenous B2M and TCR are knocked out using gene editing techniques. In some embodiments, the engineered T cells are genetically engineered to express a CS1-CAR, and endogenous CS1, B2M, and TCR are knocked out using gene editing techniques.

The application includes, for example, chinese application publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN 108884459A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176A, CN109880803A, CN 110055275A, CN110123837A, CN 110438082A, CN 110468105a international application publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018/018958A1, WO2014180306 A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A1, WO2018108106A1, WO 2018/219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/141270, WO 2019/149927, WO2019/170147A1, WO2019/210863, WO2019/219029, and methods of making such CAR-T cells.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

EXAMPLE 1 preparation of CS1 recombinant protein

(1) Construction of hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cysSLAMF 7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc expression plasmids

Primers were designed based on the sequence information of human SLAMF7 (Gene ID: 57823), mouse SLAMF7 (Gene ID: 75345), cynomolgus Monkey SLAMF7 (Gene ID: 102133710) in Genbank, and the extracellular region (SEQ ID NO: 57) of human SLAMF7 was ligated with the avi tag and His tag by molecular cloning technique to construct an hSLAMF7-avi-His expression plasmid; fusion of the human SLAMF7 extracellular region (SEQ ID NO: 57) with the human Fc fragment (SEQ ID NO: 60) to construct an hSLAMF7-huFc expression plasmid; fusing the mouse SLAMF7 extracellular region (SEQ ID NO: 58) with a human Fc fragment (SEQ ID NO: 60) to construct an mSLAMF7-huFc expression plasmid; fusion of Cynomolgus Monkey SLAMF extracellular region (SEQ ID NO: 59) with human Fc fragment (SEQ ID NO: 60) to construct cysLAMF7-huFc expression plasmid; fusing a human Ig like-V domain and a mouse Ig like-C domain, namely fusing 23 rd to 130 th (SEQ ID NO: 67) of a human SLAMF7 amino acid sequence with 128 th to 224 th (SEQ ID NO: 68) of a mouse SLAMF7 amino acid sequence to obtain a sequence (SEQ ID NO: 65), and fusing with a human Fc fragment (SEQ ID NO: 60) to construct an h1m2D-SLAMF7-huFc expression plasmid; the mouse Ig like-V domain and the human Ig like-C domain are fused, namely 23 rd to 127 th (SEQ ID NO: 69) of the mouse SLAMF7 amino acid sequence and 131 th to 226 th (SEQ ID NO: 70) of the human SLAMF7 amino acid sequence are fused to obtain a sequence (SEQ ID NO: 66), and then fused with the human Fc fragment (SEQ ID NO: 60) to construct the m1h2D-SLAMF7-huFc expression plasmid.

(2) Transfection expression and purification of recombinant proteins hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cysAMF 7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc

HEK 293F cells (from Shanghai tumor institute) well grown were transfected with the above hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cysAMF 7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc expression plasmids, 37℃5% CO ₂ The culture was continued for 7 days with a shaking table at 125rpm, centrifuged at 4000rpm for 10min, the precipitate was removed, the supernatant was collected and filtered with a 0.45 μm filter membrane, and the treated sample was purified with a HisTrap or Mabselect Sure (commercially available from GE) affinity chromatography column to finally obtain purified recombinant proteins hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cysSLAMF 7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc.

Example 2 production of anti-CS 1 hybridoma antibody

(1) Immunization

The immunogen hSLAMF7-avi-His recombinant protein and Freund's complete adjuvant 1 were selected from 6-8 week old female Balb/c mice: 1 (volume ratio), and performing primary immunization by subcutaneous multipoint injection, wherein the immunization dosage is 100 mug/person. The immunogen hSLAMF7-avi-His recombinant protein and freund's incomplete adjuvant 1:1 (volume ratio), performing subsequent immunization by subcutaneous multipoint injection, and immunizing once every two weeks for 2 times, wherein the immunization dose is 100 mug/dose. 3 days before spleen collection, boost was performed by intraperitoneal injection of 100. Mu.g/hSLAMF 7-avi-His recombinant protein alone.

(2) Hybridoma preparation

Spleen cells from immunized mice were isolated, mixed with mouse myeloma cells SP2/0 (ATCC, CRL 1581) at a ratio of 2:1, and the isolated spleen cells were fused with SP2/0 using 50% PEG (Sigma) according to published standard protocols (Kohler, milstein, 1975). After screening by HAT medium (sigma, H0262) and ELISA, flow cytometry, 3 hybridoma antibodies 32A12MAb (IgG 1/k), 37A3MAb (IgG 1/k) and 48G9MAb (IgG 2 b/k) binding to hSLAMF7 were obtained.

The sequences of the control antibodies Luc90 and huLuc63 were from patent US9175081B2, luc90 heavy chain variable region sequence (SEQ ID NO: 61), luc90 light chain variable region sequence (SEQ ID NO: 62), huLuc63 heavy chain variable region sequence (SEQ ID NO: 63), huLuc63 light chain variable region sequence (SEQ ID NO: 64); control antibodies were prepared for subsequent detection by conventional recombinant expression techniques.

TABLE 1 hybridoma antibody sequences

	Amino acid sequence	Nucleotide sequence
32A12 VH	SEQ ID NO:1	SEQ ID NO:2
32A12 VL	SEQ ID NO:6	SEQ ID NO:7
37A3 VH	SEQ ID NO:11	SEQ ID NO:12
37A3 VL	SEQ ID NO:16	SEQ ID NO:17
48G9 VH	SEQ ID NO:21	SEQ ID NO:22
48G9 VL	SEQ ID NO:26	SEQ ID NO:27

Example 3 determination of binding Activity of anti-CS 1 hybridoma antibody to recombinant protein hSLAMF7-avi-His Using ELISA method

Coating hSLAMF7-avi-his,1.5 μg/ml,100 μl/well, overnight at 4deg.C; sealing 5% skimmed milk powder PBS for 2h at room temperature; after blocking, the cells were washed with PBST, and then purified antibodies 32A12MAb, 37A3MAb, 48G9MAb were added, respectively, and diluted 11 times in 5-fold gradient from 100. Mu.g/mL, 100. Mu.L/well, and incubated at room temperature for 1 hour; after PBST wash, 100. Mu.L 4000-fold diluted HRP-coat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M 4820V) was added to each well, incubated at room temperature for 1 hour, and TMB was added after wash for development. Using enzyme labelling instrument (Spectra) 340PC 384) and OD450nM and OD650nM absorbance was determined and plotted using GraphPad prism8.0 with OD450-OD650 as the final detection result.

As shown in FIG. 1, 3 hybridoma antibodies 32A12MAb, 37A3MAb, 48G9MAb, and control antibody huLuc63 bind to the hSLAMF7-avi-his protein and exhibit concentration gradient dependence with EC50 values approaching (ranging from 0.034. Mu.g/ml to 0.058. Mu.g/ml).

Example 4 determination of binding Activity of anti-CS 1 hybridoma antibodies to human multiple myeloma cells

Taking CS1 positive MM.1S cells (human multiple myeloma cells from China academy of sciences typical culture Collection Committee cell bank) 2×10 ⁵ The individual cells/holes are placed in a 96-hole round bottom culture plate, purified antibodies 32A12MAb, 37A3MAb and 48G9MAb are respectively added after washing, and 5-time gradient dilution is carried out for 7 times at the beginning of 20 mu g/mL, 100 mu L is carried out, and the temperature is 4 ℃ for 45min; centrifuging at 500g for 5min, discarding supernatant, and removing supernatant with 1% FBSAfter 2 washes with PBS of the same type, go-anti-Mouse FITC (KANFCHEN, CAT: KC-MM-095,1:200 use) was added, 50. Mu.L/well, and incubated at 4℃for 45min;500g was centrifuged for 5min, the supernatant was discarded, washed 2 times with PBS containing 1% FBS, and 200. Mu.L of PBS containing 1% FBS was added to resuspend cells for detection by flow cytometry (Guava easy Cyte 8 HT), and the results were plotted using FlowJo7.6.1 statistics and GraphPad prism 8.0.

The results (FIG. 2) showed that the hybridoma antibodies 37A3MAb and 48G9MAb were strong in binding to MM.1S cells, the EC50 values were 0.05. Mu.g/ml and 0.18. Mu.g/ml, respectively, the hybridoma antibody 32A12MAb was weak in binding to MM.1S cells, and the EC50 value was 1.76. Mu.g/ml.

Example 5 detection of binding of anti-CS 1 hybridoma antibodies to different species of SLAMF7 recombinant proteins Using ELISA

Coating hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, hu-Fc,5 μg/mL,100 μl/well, overnight at 4deg.C; sealing 5% skimmed milk powder PBS for 2h at room temperature; after blocking, the cells were washed with PBST, and then purified antibodies 32A12MAb, 37A3MAb, 48G9MAb 5. Mu.g/mL, 100. Mu.L/well, were added, respectively, and incubated for 1 hour at room temperature; after PBST wash, 100. Mu.L 4000-fold diluted HRP-coat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M 4820V) was added to each well, incubated at room temperature for 1 hour, and TMB was added after wash for development. The absorbance values of OD450nM and OD650nM were determined using an microplate reader and OD450-OD650 was used as the final detection result.

The results (FIG. 3) show that 3 hybridoma antibodies 32A12MAb, 37A3MAb, 48G9MAb all bound to human SLAMF7-huFc, not to mouse SLAMF7-huFc, antibodies 37A3MAb and 48G9MAb bound to monkey SLAMF7-huFc, and antibody 32A12MAb did not bind to monkey SLAMF 7-huFc.

Example 6 detection of binding epitope of anti-CS 1 hybridoma antibody to hSLAMF7

Coating hSLAMF7-huFc, hu-Fc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc,5 μg/mL,100 μl/well, overnight at 4deg.C; sealing for 2 hours at room temperature; after washing, purified antibodies 32A12MAb, 37A3MAb, 48G9MAb 5. Mu.g/mL, 100. Mu.L/well were added, respectively, and incubated for 1 hour at room temperature; after washing, 100. Mu.L 4000-fold diluted HRP-coat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M 4820V) was added to each well, incubated at room temperature for 1 hour, and after washing TMB was added for development. The absorbance values of OD450nM and OD650nM were determined using an microplate reader and OD450-OD650 was used as the final detection result.

The results (Table 2) show that antibodies 37A3MAb, 48G9MAb and control antibody Luc90 bind to hCS1 with an epitope on the Ig-like V-type domain of hCS1 distal to the membrane; antibody 32A12MAb and control antibody huLuc63 bind to hCS1 epitope on the Ig-like C2-type domain of hCS1, proximal to the membrane.

TABLE 2 analysis of the binding epitope between CS1 antibody and hSLAMF7-huFc

Antibodies to	hSLAMF7-huFc	h1m2D-SLAMF7-huFc	m1h2D-SLAMF7-huFc	hu-Fc
32A12MAb	+	-	+	-
37A3MAb	+	+	-	-
48G9MAb	+	+	-	-
huLuc63	+	-	+	-
Luc90	+	+	-	-

Example 7 detection of binding of anti-CS 1 hybridoma antibodies to cells

Taking CS 1-positive human myeloma cells MM.1S (China academy of sciences typical culture Collection Committee cell Bank), NCI H929 (China academy of sciences typical culture Collection Committee cell Bank), RPMI 8226 (ATCC), respectively; and CS1 negative human embryo lung fibroblast WI38 (China academy of sciences typical culture collection committee cell bank), human embryo kidney cell HEK293 (China center for type culture collection), 2×10 ⁵ Washing 2 times after centrifuging and discarding the supernatant in a cell/well 96-well round-bottomed culture plate, and then adding 10 mug/mL of purified antibodies 32A12MAb, 37A3MAb and 48G9MAb, 100 mug and 45min at 4 ℃; centrifuging, discarding supernatant, cleaning for 2 times, adding Goat-anti-Mouse FITC (1:200), 50 μl/well, and incubating at 4deg.C for 45min; after centrifugation and washing 2 times, the resuspended cells were examined by flow cytometry (Guava easyCyte 8 HT) and the results were plotted using FlowJo7.6.1 statistics.

The results (FIG. 4) show that 3 CS1 hybridoma antibodies 32A12MAb, 37A3MAb, 48G9MAb specifically bound CS1 positive cells (MM.1S, RPMI8226, NCI-H929) and did not bind CS1 negative cells (HEK 293, WI 38).

Example 8 preparation of humanized antibodies

The hybridoma antibodies 32A12, 37A3, 48G9 were humanized by CDR grafting (Jones et al 1986). And (3) through sequence similarity comparison, selecting an antibody germline with highest similarity as an antibody template.

The germline sequence IGHV 3-7.multidot.01 from the IMGT database was selected as the antibody template for the 32A12 heavy chain, and the germline sequence IGKV 1-9.multidot.01 from the IMGT database was selected as the antibody template for the 32A12 light chain. The CDR region of the 32A12 antibody was replaced with the CDR region of the antibody template, and at the same time, threonine at position 61 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light chain (SEQ ID NO: 34) and heavy chain (SEQ ID NO: 31) of the humanized antibody hu32A 12.

The germline sequence IGHV1-69 x 02 from IMGT database was selected as antibody template for the 37A3 heavy chain, and the germline sequence IGKV1-33 x 01 from IMGT database was selected as antibody template for the 37A3 light chain. The CDR regions of the 37A3 antibody were replaced with the CDR regions of the antibody template to form the light chain (SEQ ID NO: 38) and heavy chain (SEQ ID NO: 36) of humanized antibody hu37A 3.

The germline sequence IGHV1-2 x 02 from the IMGT database was selected as the antibody template for the 48G9 heavy chain, and the germline sequence IGKV1-33 x 01 from the IMGT database was selected as the antibody template for the 48G9 light chain. The CDR region of the 48G9 antibody was replaced with the CDR region of the antibody template, and at the same time, serine at position 102 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light chain (SEQ ID NO: 43) and heavy chain (SEQ ID NO: 40) of the humanized antibody hu48G 9.

Humanized antibodies hu32a12, hu37A3, hu48G9 were prepared for subsequent detection by conventional recombinant expression techniques.

TABLE 3 sequence of humanized antibodies

	Amino acid sequence	NucleosideAcid sequence
hu32A12 VH	SEQ ID NO:31	SEQ ID NO:33
hu32A12 VL	SEQ ID NO:34	SEQ ID NO:35
hu37A3 VH	SEQ ID NO:36	SEQ ID NO:37
hu37A3 VL	SEQ ID NO:38	SEQ ID NO:39
hu48G9 VH	SEQ ID NO:40	SEQ ID NO:42
hu48G9 VL	SEQ ID NO:43	SEQ ID NO:44

EXAMPLE 9 determination of binding Activity of CS1 humanized antibody to recombinant protein hSLAMF7-avi-His Using ELISA

Coating hSLAMF7-avi-his,1.5 μg/ml,100 μl/well, overnight at 4deg.C; sealing for 2 hours at room temperature; after blocking, washing, adding humanized antibodies hu32A12, hu37A3 and hu48G9 respectively, and performing 5-time gradient dilution for 11 times from 100 mu G/mL,100 mu L/hole, and incubating at room temperature for 1 hour; after washing, 100. Mu.L 4000-fold diluted HRP-coat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M 4820V) was added to each well, incubated at room temperature for 1 hour, after washing TMB was added for color development. Using enzyme labelling instrument (Spectra)340PC 384) and OD450nM and OD650nM absorbance was determined and plotted using GraphPad prism8.0 with OD450-OD650 as the final detection result.

The results (FIG. 5) show that 3 humanized antibodies hu32A12, hu37A3, hu48G9 bind EC50 at the same level as the hSLAMF7-avi-his protein, slightly better than the control antibodies Luc90, huLuc63, and better than the corresponding murine anti-32A 12MAb, 37A3MAb, 48G9MAb.

TABLE 4 comparison of the binding of murine and humanized antibodies to the recombinant protein hSLAMF7-avi-His

EC50(μg/mL)	32A12	37A3	48G9	Luc90	huLuc63
Mouse resistance	0.0584	0.03424	0.03496	0.02117
Humanized antibodies	0.01126	0.01726	0.01576		0.022

Example 10 determination of binding Activity of CS1 humanized antibody to human multiple myeloma cells

Taking MM.1S cells 2×10 ⁵ The individual cells/wells were placed in 96-well round-bottomed plates, centrifuged to discard the supernatant, washed 2 times, and then diluted 7 times with 5-fold gradient from the beginning of adding humanized antibodies hu32A12, hu37A3, hu48G 9. Mu.g/mL, 100. Mu.L, 45min at 4℃respectively; centrifuging, discarding supernatant, washing for 2 times, adding gold-anti-Mouse FITC (KANFCHEN, CAT: KC-MM-095, 1:200), and incubating at 4deg.C for 45min; after centrifugation and washing 2 times, resuspended cells were examined by flow cytometry (Guava easyCyte 8 HT) and the results plotted using FlowJo7.6.1 statistics and GraphPad prism 8.0.

The results (FIG. 6) show that humanized antibodies hu37A3, hu48G9 bind more strongly to MM.1S, similar to the control antibody Luc 90-mFc; the humanized antibody hu32A12 was less able to bind to MM.1S, similar to the control antibody huLuc 63-mFc.

TABLE 5 comparison of the binding Capacity of murine and humanized antibodies to MM.1S

EC50(μg/mL)	32A12	37A3	48G9	Luc90	huLuc63
Mouse resistance	1.758	0.05386	0.1845	0.08235
Humanized antibodies	96.01	0.06766	0.09478		～7644

Example 11 detection of binding of CS1 humanized antibodies to different species of SLAMF7 recombinant proteins

Coating hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7, hu-Fc,5 μg/mL,100 μl/well, overnight at 4deg.C; sealing for 2 hours at room temperature; washing, then adding humanized antibodies hu32A12, hu37A3 and hu48G95 mug/mL, 100 mug/hole respectively, and incubating for 1 hour at room temperature; after washing, 100. Mu.L 4000-fold diluted HRP-coat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M 4820V) was added to each well, incubated at room temperature for 1 hour, and after washing TMB was added for development. The absorbance values of OD450nM and OD650nM were determined using an microplate reader and OD450-OD650 was used as the final detection result.

The results (fig. 7) show that both humanized antibodies hu37A3, hu48G9 bound to both human SLAMF7 and monkey SLAMF7, humanized antibody hu32a12 bound only to human SLAMF7, and none of the 3 humanized antibodies hu37A3, hu48G9, hu32a12 bound to murine SLAMF 7.

Example 12 detection of binding of CS1 humanized antibody to cells

Taking CS1 positive cells MM.1S, NCI-H929 and CS1 negative cellsWI38、HEK293，2×10 ⁵ Washing 2 times after centrifuging and discarding supernatant in a cell/well 96-well round-bottomed culture plate, and then adding humanized antibodies hu32A12, hu37A3, hu48G9 5 mug/mL, 100 mug and 45min at 4 ℃; centrifuging, discarding supernatant, cleaning for 2 times, adding Goat-anti-Mouse FITC (1:200), 50 μl/well, and incubating at 4deg.C for 45min; after centrifugation and washing 2 times, the resuspended cells were examined by flow cytometry (Guava easyCyte 8 HT) and the results were plotted using FlowJo7.6.1 statistics.

The results (FIG. 8) showed that both CS1 humanized antibodies hu37A3 and hu48G9 bound significantly to CS1 positive cells (MM.1S, NCI-H929), and that hu32A12 bound weakly to CS1 positive cells (MM.1S, NCI-H929), and none of the three humanized antibodies bound to CS1 negative cells (HEK 293, WI 38).

Example 13 determination of affinity of CS1 humanized antibody

Using Mouse Antibody Capture Kit (cat: BR 100838) from Cytiva, 3 humanized antibodies hu32A12, hu37A3, hu48G9 were each coated on CM5 chips (cat: BR 100012) by amino coupling according to the instructions. The target antibodies hu32A12, hu37A3 and hu48G9 are respectively fused with mFc (SEQ ID NO: 75) to obtain hu32A12-mFc, hu37A3-mFc and hu48G9-mFc fusion proteins which are taken as ligands, and the recombinant expressed huSLAMF7-avi-his or cysSLAMF 7-huFc takes 3-fold gradient dilution from 500nM as a mobile phase. Affinity results were obtained after detection by fitting using Evaluation Software.

The results (Table 6) show that humanized antibodies hu37A3, hu48G9 and human CS1 have affinities KD of 3.9nM (FIG. 9), 940pM (FIG. 10), respectively, which are higher than the control antibody KD7nM (FIGS. 11, 12); the humanized antibodies hu37A3, hu48G9 and monkey CS1 had affinities KD of 37nM (fig. 13) and 107nM (fig. 14), respectively, with weak affinities. Antibody hu32a12 had an affinity of 12.1nM for human CS1 (fig. 15), with weaker affinity.

TABLE 6 affinity assay results for CS1 humanized antibodies

EXAMPLE 14 analysis of aggregation of CS1 humanized antibody-Fc fusion protein

The humanized single chain antibody fusion proteins hu37A3-mFc, hu48G9-mFc, hu32A12-mFc after affinity chromatography were analyzed for aggregation by SEC. Column XK16/70 (GE Healthcare), chromatography medium 120mL Superdex200 prep grade, mobile phase PBS solution, 1mL/min flow rate equilibrate column over 1.5CV until UV280, conductivity and pH baseline plateau. The sample was loaded using a 0.5mL sample loop at a purification flow rate of 1mL/min and a monomer peak volume of about 67 mL.

The results (FIGS. 16-18) show that the monomeric antibody fractions of hu37A3-mFc, hu32A12-mFc, and hu48G9-mFc were 82%, 83%, and 93%, respectively. From the aggregation level, the monomer ratio of the 3 humanized antibodies is more than 80%, which indicates that the scFv structures of the 3 antibodies are stable and are not easy to aggregate.

Example 15: preparation of anti-CS 1 chimeric antigen receptor CAR-T cells

(1) Construction of hu32A12 scFv, hu37A3 scFv, hu48G9 scFv

VH and VL fragments of humanized antibodies hu32a12, hu37A3, hu48G9, respectively, constitute hu32a12 scFv, hu37A3 scFv, hu48G9 scFv. hu32A12 scFv amino acid sequence (SEO ID NO: 50), hu37A3 scFv amino acid sequence (SEO ID NO: 51), hu48G9 scFv amino acid sequence (SEO ID NO: 52).

(2) Preparation of chimeric antigen receptor CAR-T cells

The chimeric antigen receptor sequences shown in Table 7 were inserted into PRRRLIN-cPPT.EF-1α as vectors to construct lentiviral plasmids expressing the second-generation chimeric antigen receptors of humanized antibodies hu32A12, hu37A3, hu48G9, huLuc63, luc90, including PRRL-hu32A12 BBz, PRRL-hu37A3 BBz, PRRL-hu48G9 BBz, PRRL-huLuc63 BBz, and PRR-Luc90 BBz, respectively. Specific methods are described in the literature as (Berchovich R, 2018). The lentiviral plasmids are respectively transfected into 293T cells to obtain corresponding lentiviruses hu32A12 BBz CAR, hu37A3 BBz CAR, hu48G9 BBz CAR, huLuc63 BBz CAR and Luc90 BBz CAR.

TABLE 7 chimeric antigen receptor

Note that: CD 8. Alpha. Signal peptide sequence (SEQ ID NO: 71); scFv (hu 32A 12) sequence (SEQ ID NO: 50); scFv (hu 37A 3) sequence (SEQ ID NO: 51); scFv (hu 48G 9) sequence (SEQ ID NO: 52); huLuc63 VH sequence (SEQ ID NO: 63), (G4S) 3linker (SEQ ID NO: 74), huLuc63 VL sequence (SEQ ID NO: 64); luc90 VH sequence (SEQ ID NO: 61), luc90 VL sequence (SEQ ID NO: 62); CD 8. Alpha. Hinge region sequence (SEQ ID NO: 53); CD 8. Alpha. Transmembrane domain sequence (SEQ ID NO: 54), CD137 intracellular signaling domain sequence (SEQ ID NO: 55), CD3Zeta intracellular signaling domain sequence (SEQ ID NO: 56), hu32A12 BBz sequence (SEQ ID NO: 45), hu37A3 BBz sequence (SEQ ID NO: 46), hu48G9 BBz sequence (SEQ ID NO: 47), huLuc63 BBz sequence (SEQ ID NO: 48), luc90 BBz sequence (SEQ ID NO: 49).

T lymphocyte activation: PBMC cells were isolated from human peripheral blood to a concentration of about 1X10 ⁶ The individual cells/mL are added into lymphocyte culture medium for culture, and magnetic beads coated with anti-CD 3 and CD28 antibodies and recombinant human IL-2 with the final concentration of 300U/mL are added at 37 ℃ and 5% CO ₂ Culturing for 48h by lower stimulation;

then adding 1x10 respectively ⁷ The hu32A12 BBz CAR, hu37A3 BBz CAR, hu48G9 BBz CAR, huLuc63 BBz CAR and Luc90 BBz CAR lentiviral transduction prepared as described above gave hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells and Luc90 BBz CAR-T cells, respectively.

Example 16: t lymphocyte chimeric antigen receptor expression

The CAR-T cells prepared in example 15 were washed and suspended in flow cytometer buffer (phosphate buffer (PBS) with 1% Fetal Bovine Serum (FBS)) at a cell density of 1x10 ⁶ Individual cells/wells. CS1 antigen (10. Mu.g/mL) labeled with Biotin (Biotin) was added and incubated at 4℃for 45 min with FACS buffer. The cells were washed twice with flow cytometer buffer. The Strepitavidin-PE Conjugate antibody (eBiosciences, 1:200, diluted with FACS buffer) was added and incubated at 4deg.C for 30 min in the dark. The cells were resuspended with 300. Mu.l of flow cytometer buffer and detected with a flow cytometer.

The results show that: the CAR T cell CAR positive rates formed by antibodies hu32a12, hu37A3, hu48G9 were superior to the control antibody huluc63 (fig. 19), and were very stable with the positive rates remaining substantially unchanged over 5-11 days of culture (fig. 20).

Example 17: cytotoxicity assays for CS1 CAR T cells

The killing effect of CS1 CAR-T cells on tumor cell lines in vitro was examined by Lactate Dehydrogenase (LDH) method. By CytoToxNon-Radioactive Cytotoxicity Assay kit (Promega, G1780) for detection of cytotoxicity, specific procedures are described in the kit instructions.

Target cells: CS 1-expressing multiple myeloma cells MM.1S, NCl-H929, RPMI-8226 and HEK293 cells that do not express CS 1.

Effector cells: UTD (non-virus infected T cells), hu32a12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells.

Effector cells and target cells (10000 cells/well) were sequentially plated into 96-well plates (200ul 1640+10%FBS system per well) at 37℃and 5% CO at an effective target ratio of 3:1,1:1,1:3, respectively ₂ The following co-cultivation was performed for 16 hours while spontaneous release wells and maximum release wells were set according to the instructions. First, the20 μl of lysate was added to the maximum release wells for the next day and reacted for 30 minutes. Then, 50. Mu.l of the culture supernatant was removed from each well, and 50. Mu.l of the substrate was added thereto, and incubated at room temperature for 25 minutes in the absence of light, and 50. Mu.l of the stop solution was added thereto to terminate the reaction, and the absorbance peak at OD490/650nm of each well was detected by an ELISA reader.

The kill rate was calculated according to the following formula:

cytotoxicity% = [ experimental group LDH release amount (avg.) -effector cell spontaneous LDH release amount (avg.) -target cell spontaneous LDH release amount (avg.) ]/[ target cell maximum LDH release amount (avg.) -target cell spontaneous LDH release amount (avg.) -volume calibration (avg.) ] ×100%

The results show that: the hu32A12 BBz CAR-T cells, the hu37A3 BBz CAR-T cells and the hu48G9 BBz CAR-T cells have obvious in-vitro killing effect on target cells expressing CS1, and have obvious dose-response relation with the effective target ratio. For HEK293 cells that did not express CS1, CS1 CAR T had little killing effect at different potency target ratios (fig. 21). It is demonstrated that hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells and hu48G9 BBz CAR-T cells can specifically recognize and kill CS1 positive tumor cells.

Example 18: CS1 CAR T cell in vitro induced cytokine release experiment

The hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells of example 17 were further examined for cytokine secretion under target cell stimulation.

Supernatants of hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells and tumor cells of example 17 were collected and incubated as BD ^TM The CBA kit (BD Pharmingen Co.) was used for cytokine detection.

The results show that: secretion of cytokine IFN-gamma: after hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells were co-incubated with MM.1S cells, secretion of IFN-gamma cytokines was activated and a dose-response relationship was exhibited with the effective target ratio (FIG. 22).

Secretion of the cytokine TNF- α: secretion of each CS1 CAR-T TNF- α was significantly enhanced after co-incubation of hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells with MM.1S cells (FIG. 23). After co-incubation with HEK293 cells not expressing CS1, none of the CS1 CAR T had significant TNF- α secretion, indicating that it did not produce non-specific TNF- α cytokine secretion.

Secretion of cytokine IL-2: after co-incubation of hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, luc90 BBz CAR-T cells with MM.1S cells, the secretion of IL-2 correlated inversely with the effective target ratio, which was likely related to the consumption of IL-2 by T cell proliferation, the lower the effective target ratio, the higher the concentration of IL-2 (FIG. 24). After co-incubation with HEK293 cells not expressing CS1, no significant IL-2 secretion was seen for each CS1 CAR-T, indicating that no non-specific IL-2 secretion was generated after co-incubation of CS1 CAR-T with CS1 negative cells.

Example 19: effect of soluble CS1 protein on in vitro cytotoxic killing of CS1 CAR T cells

In clinical studies, soluble CS1 protein was found to be present in patients with Multiple Myeloma (MM), and literature (Marko Ishibashi, 2018) indicated that the concentration of soluble CS1 in the serum of MM patients was mostly 0.091-14.7ng/mL. Therefore, we detected the effect of CAR-T cytotoxic killing by CS1 CAR by adding different concentrations of recombinantly expressed CS1 protein (0, 1, 10, 100 ng/mL) immediately after the addition of effector and target cells, based on the detection of CAR-T cytotoxic killing by LDH method of example 17.

The results showed that the killing effect of CS1 CAR-T cells on target cells did not significantly change with increasing concentration of soluble CS1 protein (fig. 25), indicating that soluble CS1 did not affect CS1 CAR-T cytotoxic killing in vitro.

Example 20: determination of expression of depletion markers of CS1 CAR T cells stimulated by target antigen

Detection of CS1 CAR-T by flow after stimulation with target antigen CS 1-positive multiple myeloma cell line NCl-H929, markers PD-1, TIM-3 were depletedExpression of LAG-3. The specific process is as follows: CS 1-positive multiple myeloma cell line NCl-H929 (50000 cells/well, 500. Mu.l/well) was added to each CS1 CAR-T (50000 cells/well, 500. Mu.l/well) in 48-well cell culture plates, and UTD (non-CAR transfected T cells) was set up simultaneously as a control. At 37℃with 5% CO ₂ And (5) standing and culturing in an incubator for 48 hours. Cells were harvested by centrifugation of 48-well plates. Cells were washed and transferred to 96-well plates. Two groups were separated, one group was added with 50. Mu.l/well PD-1 (BD HorizonTM, 562516)/TIM-3 (BD PharmingenTM, 563422)/CD 3 (Invitrogen, 17-0028-42), and the other group was added with LAG-3 (BD HorizonTM, 565720)/CD 3 (BD, 1:100, diluted with FACS buffer) antibody mix solution, and incubated at 4℃for 30 minutes in the absence of light.

Cells were washed, resuspended in 300 μl FACS buffer for each sample, and detected by flow cytometry.

The results show that: depletion marker PD-1: expression of each CS1 CAR-T PD-1 was significantly up-regulated after target cell stimulation compared to UTD, with the provided PD-1 expression levels of CS1-CAR T of the application being lower than the PD-1 expression levels of control luc90 CAR T (fig. 26).

Depletion of marker Tim-3 expression of each CS1 CAR-T Tim-3 was significantly up-regulated after stimulation with target cells compared to UTD, wherein the levels of Tim-3 expression of CS1-CAR T provided by the present application were lower than control luc90 CAR T (fig. 27).

Depletion marker LAG-3: expression of each CS1 CAR-T LAG-3 was significantly up-regulated after target cell stimulation compared to UTD, with levels of LAG-3 expression of hu37A3 CAR-T and hu48G9 CAR-T being lower than control luc90 CAR T (FIG. 28).

Taken together, CS1 CAR-T had weak expression of the hu32a12 CAR-T, hu37A3 CAR-T, hu48G9 CAR-T group depletion markers following stimulation by target antigen CS1 positive multiple myeloma cells. Indicating that hu32A12 CAR-T, hu37A3 CAR-T, hu48G9 CAR-T less entered the depleted state after incubation with tumor cells.

Example 21: CS1 CAR-T CD 3-zeta phosphorylation autoactivation (TONIC signaling) study

Detecting the phosphorylation proportion of CD 3-zeta in CS1 CAR-T cell by using a conventional molecular biology technology Western-blot method, and judging the activation condition of an antigen independent signal transduction pathway.

Taking cell lysate to carry out SDS gel electrophoresis, membrane transfer and sealing. Antibody staining: after overnight washing with CD 3-zeta and p-CD 3-zeta antibody solutions incubated with membranes, goat anti-mouse-HRP secondary antibody (1:5000) was incubated, and chemiluminescent and light exposure assays were performed after 1 hour at room temperature (FIG. 29). The results (fig. 29) demonstrate that, when hu32a12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells were routinely cultured in vitro to day 9, the extent of CD3- ζ phosphorylation was significantly weaker than the control huluc63 CAR T cells and luc90 CAR T cells, indicating that hu32a12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells had fewer self-activation during in vitro culture without target cell stimulation. It is predicted that the self-depletion of hu32A12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells is less.

Example 22: measurement of IL-6 secretion after Co-incubation of CS1 CAR T cells with monocytes and tumor cells

Co-culturing CS1 CAR-T with monocytes and CS1 positive multiple myeloma cells NCl-H929 at a ratio of cell number ratio of 1:1:1 for 48 hr, collecting culture supernatant, and using BD ^TM The CBA kit performs cytokine detection according to the instructions.

The results are shown in FIG. 30, where the cytokine IL-6 was secreted after incubation of CAR-T with monocytes and target cells. hu32A12 CAR-T, hu37A3 CAR-T, hu48G9 CAR-T cells secreted less IL-6.

Example 23: CS1 CAR T cell in vitro specific amplification test

The experiment uses T cells transduced with CS1-CAR and T cells not transduced with CAR (UTD) as effector cells, and the effector cells are respectively incubated with CS1 positive target cells MM.1S, NCl-H929, RPMI-8226 cells and CS1 negative target cells HEK293, wherein the effective target ratio is 2:1. the total cell number was then counted every three days and examined for CD3 positive rate and PI staining activity for 9 days.

The results showed that when huIL-2 was not added to the culture medium, hu37A3 CAR-T, hu48G9 CAR-T, huluc63 CAR-T, luc90 CAR-T showed significant specific proliferation of CAR-T cells (FIG. 31) and significant increase in the viability of CAR-T cells (PI staining identification) after co-incubation with CS1 positive target cells, respectively (FIG. 32). At the same time, UTD cells proliferated slowly after co-incubation with all target cells (fig. 33). In the positive control group with 300IU/mL huIL-2 added in the culture medium and no target cells, UTD, hu37A3 CAR-T and hu48G9 CAR-T cells all had sustained proliferation, and the activity rate was maintained at 90% or more (FIG. 34).

The results show that hu37A3 CAR-T, hu48G9 CAR-T cells produced significant specific proliferation after co-incubation with CS1 positive multiple myeloma cells.

Example 24: cell killing experiments of CS1 CAR T cells in mice

(1) CS1 CAR T cell killing myeloma cell MM.1S experiments in mice

Inoculation of 3X 10 ⁶ Human multiple myeloma cells mm.1s were 5-6 weeks old, female NPG mice (from beijing verda biotechnology limited) were subcutaneously on the right axilla, and tumor vaccinated diary was D0. When the tumor volume is as long as about 180mm ³ Randomly divided into 6 groups, namely UTD control group, hu37A3 CAR-T, hu G9 CAR T cell treatment group, and corresponding CAR T cells were injected into tail vein at a dose of 4×10 ⁶ Individual cells/mice.

The experimental results showed that at D38, the tumor inhibition rates for each group were hu37A3 CAR-T, respectively, compared to UTD: 99.9% (5 mice with 4 tumor regressions), hu48G9 CAR-T:100% (total tumor regression in 5 mice). From the results, both hu37A3 CAR T group and hu48G9 CAR T group were significantly tumor-inhibiting.

(2) Anti-tumor effect of CS1 CAR T cells on subcutaneous tumor inhibition of NPG mice of human multiple myeloma cell RPMI-8226-CS1

The human SLAMF7 extracellular domain (SEQ ID NO: 57) is transferred into RPMI-8226 cells by a slow virus mediated mode by adopting a conventional molecular biology technology to construct an RPMI-8226 stable transfer cell strain RPMI-8226-CS1 which overexpresses CS1.

Inoculation of 5X 10 ⁶ Human multiple myeloma cell RPMI-8226-CS1 overexpressing CS1 was subcutaneous, swelling on the right axilla of female NPG mice (from Beijing Vietnam Biotechnology Co., ltd.) at 5-6 weeks of ageTumor inoculation diary was D0. When the tumor volume is as long as about 110mm ³ The random groups were 7, respectively:

group1: dosage of 2X 10 ⁶ UTD group; group2: dosage is 0.6X10 ⁶ hu37A3 CAR-T group; group3: dosage 1X 10 ⁶ hu37A3 CAR-T group; group4: dosage of 2X 10 ⁶ hu37A3 CAR-T group; group5: dosage is 0.6X10 ⁶ hu48G9 CAR-T group; group6: dosage 1X 10 ⁶ hu48G9 CAR-T group; group7: dosage of 2X 10 ⁶ hu48G9 CAR-T group.

The tail vein was injected with corresponding CAR T cells, body weight was measured 2 times per week (including the day of group administration and euthanasia) after injection, and tumor long and short diameters were measured and recorded with vernier calipers, tumor volumes were calculated, tumor growth curves were plotted from tumor volumes, and differences in tumor growth curves between groups were compared (tumor volumes: v=1/2×long diameter×short diameter ² )。

The experimental results show that low dose (0.6X10) ⁶ And 1X 10 ⁶ ) The hu48G9 CAR-T, hu37A3 CAR-T cells of (a) all significantly inhibited tumor growth (fig. 36).

Example 25: preparation of CS1-UCAR-T cells and CS 1-knockout UCAR-T cells

After in vitro expansion of hu48G9 CAR-T cells for 48 hours, TRAC and B2M double gene knockout or three gene knockout (TRAC, B2M and CS1 knockout) is respectively carried out on the hu48G9 CAR-T cells according to the conventional molecular biology CRISPR/Cas9 technology, so as to obtain hu48G9-UCAR-T cells (TRAC and B2M double gene knockout) and hu48G9-UCAR-CS1-/-T cells (TCR, B2M, CS1 three gene knockout). According to the instructions for the reagents (GeneArt) ^TM Precision gRNA Synthesis Kit, thermo Tisher) in vitro to synthesize a gRNA sequence targeting TRAC, B2M, CS1, wherein the nucleic acid sequence of TRAC-gRNA is as set forth in SEQ ID NO:76, the nucleic acid sequence of the B2M-gRNA is shown in SEQ ID NO:84, the nucleic acid sequence of CS1 gRNA is shown as SEQ ID NO: 89.

Example 26: anti-tumor effect of CS1-UCAR-T cells and CS1-UCAR-CS1-/-T cells on NPG mice with human multiple myeloma cells RPMI 8226-CS1 in subcutaneous transplantation

Inoculation of 5X 10 ⁶ Human multiple myeloma cells RPMI-8226-CS1 overexpressing CS1 were D0 tumor vaccinated in female NPG mice (from Beijing Vetong Biotechnology Co., ltd.) at 5-6 weeks of age. When the tumor volume is as long as about 155mm ³ CAR T cells were vaccinated and mice were randomly divided into 3 groups:

group1: dosage 1X 10 ⁶ UTD group; group2: dosage 1X 10 ⁶ hu48G9-UCAR-T group; group3: dosage 1X 10 ⁶ hu48G9-UCAR-CS1-/-T group;

Experimental results showed that mice were normal in weight by D25 days after CAR T injection. Compared with UTD group, the tumor inhibition rate of hu48G9-UCAR-T group is 99.37%, and the tumor inhibition rate of hu48G9-UCAR-CS1-/-T group is 96.49%. Demonstrating that CS 1-targeted universal CAR-T cells all significantly inhibited tumor growth (fig. 37).

In addition, the CS 1-targeted CAR-T cell is also detected to obviously resist the killing effect of NK cells on the targeted tumor antigen CAR-T or the universal CAR-T cell, and improve the persistence and/or the transplantation survival rate of autologous or allogeneic immune cells in the presence of host immune cells, so that the antitumor activity of the targeted tumor antigen CAR-T or the universal CAR-T cell is improved.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each was individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

The sequences involved in the present application are as follows:

Claims

a CS 1-targeting antigen binding unit, wherein the antigen binding unit is selected from the group consisting of:

(1) An antigen binding unit comprising a heavy chain variable region comprising HCDR1 as shown in SEQ ID No. 3, 13 or 23, and/or comprising HCDR2 as shown in SEQ ID No. 4, 14, 24 or 32, and/or comprising HCDR3 as shown in SEQ ID No. 5, 15, 25 or 41;

(2) An antigen binding unit comprising a light chain variable region comprising LCDR1 as set forth in SEQ ID No. 8, 18 or 28, and/or comprising LCDR2 as set forth in SEQ ID No. 9, 19 or 29, and/or comprising LCDR3 as set forth in SEQ ID No. 10, 20 or 30;

(3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit;

(4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antigen binding unit of any one of (1) to (3).
The antigen-binding unit of claim 1, wherein the antigen-binding unit is selected from the group consisting of:

(1) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 4, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; or alternatively

(2) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 13, HCDR2 shown in SEQ ID NO. 14, HCDR3 shown in SEQ ID NO. 15, LCDR1 shown in SEQ ID NO. 18, LCDR2 shown in SEQ ID NO. 19, LCDR3 shown in SEQ ID NO. 20; or alternatively

(3) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 25, and LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30; or alternatively

(4) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 5, LCDR1 shown in SEQ ID NO. 8, LCDR2 shown in SEQ ID NO. 9, LCDR3 shown in SEQ ID NO. 10; or alternatively

(5) An antigen binding unit comprising HCDR1 shown in SEQ ID NO. 23, HCDR2 shown in SEQ ID NO. 24, HCDR3 shown in SEQ ID NO. 41, and LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, LCDR3 shown in SEQ ID NO. 30;

(6) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (5), having the same or similar activity as the antigen binding unit of any one of (1) to (5).
The antigen-binding unit of claim 1, wherein the antigen-binding unit is selected from the group consisting of:

(1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO: 1. 11, 21, 31, 36 or 40;

(2) An antigen binding unit, said antigen binding unit having a light chain variable region having the amino acid sequence of SEQ ID NO: 6. 16, 26, 34, 38 or 43;

(3) An antigen binding unit comprising (1) a heavy chain variable region of the antigen binding unit and (2) a light chain variable region of the antigen binding unit;

(4) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (3), and having the same or similar activity as the antibody of any one of (1) to (3).
An antigen-binding unit as claimed in claim 3, wherein the antigen-binding unit is selected from the group consisting of:

(1) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:6, an amino acid sequence shown in figure 6;

(2) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:11 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:16, and a polypeptide comprising the amino acid sequence shown in seq id no;

(3) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no;

(4) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no;

(5) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:38, and a nucleotide sequence shown in seq id no;

(6) An antigen binding unit, the heavy chain variable region of said antigen binding unit having the amino acid sequence of SEQ ID NO:40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO:43, an amino acid sequence shown in seq id no;

(7) An antigen binding unit, a variant of the antigen binding unit of any one of (1) to (6), having the same or similar activity as the antigen binding unit of any one of (1) to (6).
An antigen binding unit that recognizes the same epitope as the antigen binding unit of any one of claims 1-4; or Ig-like V-type domain binding CS1 protein; or Ig-like C2-type domain binding CS1 protein.
The antigen-binding unit of any one of claims 1-5, wherein the antigen-binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody, or a fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is a fully anti-scFv, fv fragment, fab 'fragment, (Fab') ₂ Fragments, fd fragments, dAb fragments, single domain antibodies, multifunctional antibodies or scFv-Fc antibodies.
An immunological conjugate, said immunological conjugate comprising: an antigen binding unit as claimed in any one of claims 1 to 6; and a functional molecule attached thereto, the functional molecule selected from the group consisting of: molecules targeting tumor surface markers, molecules inhibiting tumors, molecules targeting surface markers of immune cells or detectable markers.
A chimeric receptor, wherein the extracellular domain of the chimeric receptor comprises the antigen binding unit of any one of claims 1-6, the chimeric receptor comprising: chimeric Antigen Receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), or a combination thereof.
The chimeric receptor according to claim 8, wherein the chimeric receptor comprises, in sequential linkage: an antigen binding unit, a transmembrane region and an intracellular signaling region according to any one of claims 1 to 6.
The chimeric receptor of claim 9, wherein the intracellular signaling region is selected from the group consisting of: CD3 ζ, fceriγ, CD27, CD28, CD137, CD134, myD88, intracellular signal region sequences of CD40, or combinations thereof; and/or the transmembrane region comprises a transmembrane region of CD8 or CD 28.
The chimeric receptor according to claim 10, wherein the chimeric receptor comprises:

the antigen binding unit, the transmembrane region of CD8/CD28, and cd3ζ of any one of claims 1-6; or (b)

The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, the intracellular signaling region of CD137, and cd3ζ; or (b)

The antigen binding unit of any one of claims 1-6, a transmembrane region of CD8/CD28, an intracellular signaling region of CD28, and cd3ζ; or (b)

The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, the intracellular signaling region of CD28, CD137 and CD3 ζ.
The chimeric receptor according to claim 11, wherein the amino acid sequence of the chimeric receptor is as set forth in SEQ ID NO: 45. 46 or 47.
Nucleic acid encoding the antigen binding unit of any one of claims 1-6, the immunoconjugate of claim 7, the chimeric receptor of any one of claims 8-12.
An expression vector comprising the nucleic acid of claim 13.
A virus comprising the expression vector of claim 14.
A composition comprising an antigen binding unit according to any one of claims 1-6, an immunoconjugate according to claim 7, and/or a chimeric receptor according to any one of claims 8-12, characterized in that the composition is cytotoxic to cells expressing CS 1.
The composition of claim 16, wherein the CS1 expressing cells are tumor cells and/or pathogen cells.
A host cell comprising the expression vector or genome of claim 14 having integrated therein the nucleic acid of claim 13.
The host cell of claim 18, which expresses the chimeric receptor of any one of claims 8-12.
The host cell of claim 18 or 19, wherein the host cell comprises a T cell, a cytotoxic T lymphocyte, an NK cell, an NKT cell, a DNT cell, a regulatory T cell, an NK92 cell, a stem cell derived immune effector cell, or a combination thereof.
The host cell of claim 20, wherein the T cell is a naturally derived T cell and/or a T cell induced by a pluripotent stem cell;

Preferably, the T cells are autologous or allogeneic T cells;

preferably, the T cell is a primary T cell;

preferably, the T cells are derived from autologous human T cells.
The host cell of claim 20 or 21, wherein the T cell comprises a memory stem cell-like T cell (Tscm cell), a central memory T cell (Tcm), an effector T cell (Tef), a regulatory T cell (Tregs), an effector memory T cell (Tem), an αβ T cell, a γδ T cell, or a combination thereof.
The host cell of any one of claims 18-22, wherein the cell comprises:

knock-out of the gene encoding the TCR protein and/or low or no expression of endogenous TCR molecules, and/or

Knockout of genes encoding MHC proteins and/or endogenous MHC low expression or non-expression.
The host cell of claim 23, wherein the endogenous MHC molecule B2M and the endogenous TCR are knocked out using CRISPR/Cas9 technology.
A host cell according to claim 24, wherein the gRNA used to knock out B2M comprises the sequences set forth in SEQ ID NOs 84, 85, 86 and/or 87 and the gRNA used to knock out TCR comprises the sequences set forth in SEQ ID NOs 76, 77, 78, 79, 80, 81, 82 and/or 83.
The host cell of any one of claims 18-25, wherein the cell comprises a knockout of a gene encoding a CS1 protein and/or low or no expression of an endogenous CS1 molecule.
The host cell of claim 26, wherein the cellular CS1 gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the group consisting of the sequences set forth in SEQ ID NOs 88, 89, 90, 91, 92, 93, 94 and/or 95.
The host cell of any one of claims 18-27, wherein the host cell binds to cells that express CS1 without significantly binding to cells that do not express CS 1.
The host cell of any one of claims 18-28, further carrying a coding sequence for an exogenous cytokine; or (b)

It also expresses another chimeric receptor; or (b)

It also expresses chemokine receptors; or (b)

It also expresses a safety switch.
A combination of an antigen binding unit according to any one of claims 1 to 6, an immunoconjugate according to claim 7, a chimeric receptor according to any one of claims 8 to 12, a composition according to claim 16 or 17, a host cell according to any one of claims 18 to 29, in combination with an agent that enhances its function, preferably in combination with a chemotherapeutic agent;

And/or in combination with an agent that ameliorates one or more side effects associated therewith;

and/or in combination with host cells expressing chimeric receptors targeted beyond CS 1.
A method of making the antigen binding unit of any one of claims 1-6, the immunoconjugate of claim 7, the chimeric receptor of any one of claims 8-12, and/or the composition of claim 16 or 17, the method comprising culturing the host cell of any one of claims 18-29 under conditions suitable for expression of the antigen binding unit, immunoconjugate, chimeric receptor, and/or composition, and isolating the antigen binding unit, immunoconjugate, chimeric receptor, and/or composition expressed by the host cell.
A pharmaceutical composition, comprising:

the antigen binding unit or nucleic acid encoding the antigen binding unit of any one of claims 1-6; or (b)

The immunological conjugate of claim 7 or a nucleic acid encoding the conjugate; or (b)

The chimeric receptor or the nucleic acid encoding the chimeric receptor of any one of claims 8-12; or (b)

The host cell of any one of claims 18-29;

and optionally, a pharmaceutically acceptable carrier or excipient.
A method of treating/diagnosing a disease comprising administering to a subject in need thereof an effective amount of an antigen binding unit of any one of claims 1-6, or an immunoconjugate of claim 7, or a host cell of any one of claims 18-29, or a composition of claim 32;

preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; preferably the tumour is multiple myeloma;

preferably, the subject is a human;

preferably, wherein the host cell is an autologous or allogeneic T cell to the subject.
Use of an antigen binding unit according to any one of claims 1 to 6, or an immunoconjugate according to claim 7, or a host cell according to any one of claims 18 to 29, or a composition according to claim 32, for the treatment and/or diagnosis of a disease, wherein the disease expresses CS1; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors, preferably the tumor is multiple myeloma.
Use of an antigen binding unit according to any one of claims 1-6, or an immunoconjugate according to claim 7, or a host cell according to any one of claims 18-29, or a composition according to claim 32, for the preparation of a medicament for killing NK cells.
Use according to claim 35, wherein the persistence and/or the survival rate of transplantation of autologous or allogeneic immune cells in the presence of host immune cells is increased.