CN117730145A - chimeric antigen receptor - Google Patents

Abstract

Provided are CAR-T cells and CARs that target NKG2A and tumor antigens simultaneously, the CARs comprising a bispecific antigen binding domain comprising a NKG2A antigen binding domain and a tumor antigen binding domain.

Description

chimeric antigen receptor

Related applications

This patent application requires the Chinese patent application with application number 202110807344.3 submitted on July 16, 2021, the Chinese patent application with application number 202110918700.9 submitted on August 11, 2021, and the Chinese patent application submitted on April 26, 2022 The priority of the Chinese patent application with application number 202210447726.4.

Sequence listing file submitted at the same time

The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (name: FF00640PCT-sequence listing.xml, date: 20220714, size: 55KB).

Technical field

This application provides chimeric antigen receptors (CARs) and engineered dual-targeting T cells and preparation methods thereof. The engineered dual-targeting T cells and compositions containing the engineered dual-targeting T cells can be used for disease treatment.

Background technique

Immune cell therapy, as a cell-based cancer therapy, is currently the most promising treatment method. Immune cells have the potential to target tumor cells while sparing normal tissue; clinical observations indicate that they have major anti-cancer activity. There remains an urgent need to improve immune cell therapies by broadening their applicability and enhancing their efficacy. The effective activation of CAR-T cells depends heavily on the specificity of the antibody that recognizes the tumor-associated antigen and the affinity of the antigen binding. Therefore, under the current situation that the design of the intracellular signal transduction region of CAR-T cells has become mature, the design of the antigen-binding region has become the focus and key to the development of new CAR-T technology.

Contents of the invention

The purpose of this application is to provide an engineered dual-targeting T cell and a chimeric antigen receptor (CAR), in which antibody fragments targeting two antigens respectively are expressed in tandem on a chimeric antigen receptor (CAR). In some embodiments, provided engineered cells exhibit enhanced immune responses, including treatment of cancer, microbial infections, and/or viral infections in a subject. In some embodiments, the engineered cells provided have both resistance to host immune rejection and anti-tumor effects. Also provided are methods of treating a subject by administering a therapeutically effective amount of the provided engineered T cells.

In one aspect, the present application provides a chimeric antigen receptor (CAR), the CAR comprising a bispecific antigen binding domain, the bispecific antigen binding domain including an NKG2A antigen binding domain and a tumor antigen binding domain, the The NKG2A antigen binding domain includes a first binding region L and a first binding region H. The tumor antigen binding domain includes a second binding region L and a second binding region H. The first binding region L, the first binding region H, The second bonding area L and the second bonding area H are connected to form a loop structure.

In some embodiments, the loop structure is formed in the following manner: the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the Connect both ends of the antibody. In certain embodiments, the antibody sequentially includes from N-terminus to C-terminus:

The binding area H, the binding area L.

In certain embodiments, the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.

In certain embodiments, the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.

In certain embodiments, the binding region H connected to the antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the binding region L of the other antigen-binding domain is the second binding region L, and the binding region H of the other antigen-binding domain is the second binding region H.

In certain embodiments, the binding region H connected to the antibody is the second binding region H, and the binding region L connected to the antibody is the second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H.

In certain embodiments, the antibody sequentially includes from N-terminus to C-terminus:

The binding area L, the binding area H.

In certain embodiments, the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.

In certain embodiments, the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.

In certain embodiments, the binding region H connected to the antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the binding region L of the other antigen-binding domain is the second binding region L, and the binding region H of the other antigen-binding domain is the second binding region H.

In certain embodiments, the binding region H connected to the antibody is the second binding region H, and the binding region L connected to the antibody is the second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H.

In certain embodiments, the first binding region L includes a first light chain or a first light chain variable region (VL), and the first binding region H includes a first heavy chain or a first heavy chain variable region. region (VH), the second binding region L includes a second light chain or a second light chain variable region (VL), and the second binding region H includes a second heavy chain or a second heavy chain variable region ( VH).

In certain embodiments, the first binding region L includes a first light chain variable region (VL), the first binding region H includes a first heavy chain variable region (VH), and the second binding region Region L contains a second light chain variable domain (VL) and the second binding domain H contains a second heavy chain variable domain (VH).

In certain embodiments, the antibody comprises a scFv.

In certain embodiments, the first light chain includes the first light chain variable region (VL), the first heavy chain includes the first heavy chain variable region (VH), and the first heavy chain variable region (VH) The two light chains comprise the second light chain variable domain (VL) and the second heavy chain comprises the second heavy chain variable domain (VH).

In certain embodiments, the NKG2A antigen binding domain comprises a single domain antibody, and/or the tumor antigen binding domain comprises a single domain antibody.

In certain embodiments, the first light chain variable region (VL) comprises a first light chain complementarity determining region (LCDR) as set forth in any one of SEQ ID NOs: 6-8, or a combination thereof.

In certain embodiments, the first heavy chain variable region (VH) comprises a first heavy chain complementarity determining region (HCDR) as set forth in any one of SEQ ID NOs: 3-5, or a combination thereof.

In certain embodiments, the first light chain variable region (VL) comprises first light chain complementarity determining region 1 (LCDR1), first light chain complementarity determining region 2 (LCDR2), first light chain complementarity determining region 2 (LCDR2), first light chain complementarity determining region 1 (LCDR1), first light chain complementarity determining region 2 (LCDR2), Region 3 (LCDR3), the first LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, the first LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and the first LCDR3 includes the amino acid sequence shown in SEQ ID NO:7 The amino acid sequence shown in ID NO:8.

In certain embodiments, the first heavy chain variable region (VH) comprises first heavy chain complementarity determining region 1 (HCDR1), first heavy chain complementarity determining region 2 (HCDR2), first heavy chain complementarity determining region 2 (HCDR2), first heavy chain complementarity determining region 1 (HCDR1), first heavy chain complementarity determining region 2 (HCDR2), Region 3 (HCDR3), the first HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, the first HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and the first HCDR3 includes the amino acid sequence shown in SEQ ID NO:3 The amino acid sequence shown in ID NO:5.

In certain embodiments, the NKG2A antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 3-8. In certain embodiments, the first heavy chain variable region (VH) comprises the amino acid sequence set forth in SEQ ID NO: 1 and the first light chain variable region (VL) comprises the amino acid sequence set forth in SEQ ID NO: 1 The amino acid sequence shown in 2.

In certain embodiments, the tumor antigens include CD19, GPC3, Claudin18.2, WT1, HER2, EGFR, BCMA, or combinations thereof.

In certain embodiments, the tumor antigen includes BCMA.

In certain embodiments, the tumor antigen binding domain includes a BCMA antigen binding domain.

In certain embodiments, the second light chain variable region (VL) comprises a second light chain complementarity determining region (LCDR) as set forth in any one of SEQ ID NOs: 12-14, or a combination thereof.

In certain embodiments, the second heavy chain variable region (VH) comprises a second heavy chain complementarity determining region (HCDR) as set forth in any one of SEQ ID NOs: 9-11, or a combination thereof.

In certain embodiments, the second light chain variable region (VL) comprises a second light chain complementarity-determining region 1 (LCDR1), a second light chain complementarity-determining region 2 (LCDR2), a second light chain complementarity-determining region Region 3 (LCDR3), the second LCDR1 includes the amino acid sequence shown in SEQ ID NO:12, the second LCDR2 includes the amino acid sequence shown in SEQ ID NO:13, and the second LCDR3 includes the amino acid sequence shown in SEQ ID The amino acid sequence shown in NO:14.

In certain embodiments, the second heavy chain variable region (VH) comprises second heavy chain complementarity determining region 1 (HCDR1), second heavy chain complementarity determining region 2 (HCDR2), second heavy chain complementarity determining region 2 (HCDR2), second heavy chain complementarity determining region Region 3 (HCDR3), the second HCDR1 includes the amino acid sequence shown in SEQ ID NO:9, the second HCDR2 includes the amino acid sequence shown in SEQ ID NO:10, and the second HCDR3 includes the amino acid sequence shown in SEQ ID NO :The amino acid sequence shown in 11.

In certain embodiments, the BCMA antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 9-14. In certain embodiments, the NKG2A antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO:3-8, and the BCMA antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO:9-14.

In certain embodiments, the second heavy chain variable region (VH) comprises the amino acid sequence set forth in SEQ ID NO: 15, and the second light chain variable region (VL) comprises the amino acid sequence set forth in SEQ ID NO: 15. The amino acid sequence shown in 16.

In certain embodiments, the second binding region L comprises a light chain variable region in the sequence shown in any one of SEQ ID NO:46-50, and the second binding region H comprises a light chain variable region as shown in any one of SEQ ID NO:46-50 : The heavy chain variable region in the sequence shown in any one of 46-50. In certain embodiments, the NKG2A antigen binding domain comprises a first heavy chain variable region as set forth in SEQ ID NO: 1 and a first light chain variable region as set forth in SEQ ID NO: 2, said The BCMA antigen binding domain includes a second heavy chain variable region as shown in SEQ ID NO:15 and a second light chain variable region as shown in SEQ ID NO:16.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

BCMA VL—NKG2A VH—NKG2A VL—BCMA VH.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

NKG2A V—BCMA VH—BCMA VL—NKG2A VH.

In certain embodiments, the connection includes connection through a linker, the linker includes Lin1 or (G4S)n, where n is an integer equal to or greater than 1; the Lin1 includes as shown in SEQ ID NO: 18 The amino acid sequence shown.

In certain embodiments, in the (G4S)n, n is 1 or 3.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

BCMA VL—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VH.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

NKG2A VL—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VH.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

BCMA VL—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VH.

In certain embodiments, the bispecific antigen-binding domain includes the following connection from the N-terminus to the C-terminus:

NKG2A VL—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VH.

In certain embodiments, the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-41.

In certain embodiments, the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 46-50.

In certain embodiments, the bispecific antigen binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20, 23, and 26.

In certain embodiments, the CAR comprises a transmembrane domain.

In certain embodiments, the CAR comprises an intracellular signaling domain.

In certain embodiments, the CAR includes a transmembrane domain and an intracellular signaling domain.

In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain.

In certain embodiments, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:30, and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:31.

In certain embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain.

In certain embodiments, the CD3ζ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:34.

In certain embodiments, the CAR further comprises a costimulatory signaling domain.

In certain embodiments, the CAR contains 2 identical or different costimulatory signaling domains.

In certain embodiments, the costimulatory signaling domain comprises a 4-1BB costimulatory signaling domain and/or a CD28 costimulatory signaling domain.

In certain embodiments, the CAR comprises a 4-1BB costimulatory signaling domain and a CD3ζ intracellular signaling domain.

In certain embodiments, the 4-1BB costimulatory signal domain includes the amino acid sequence shown in SEQ ID NO:33, and the CD28 costimulatory signal domain includes the amino acid sequence shown in SEQ ID NO:32 .

In certain embodiments, the CAR includes a hinge region.

In certain embodiments, the hinge region comprises a CD8 hinge region.

In certain embodiments, the CD8 hinge region comprises the amino acid sequence set forth in SEQ ID NO:35.

In certain embodiments, the CAR comprises a CD8 transmembrane domain and a CD8 hinge domain.

In certain embodiments, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:30, and the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO:35.

In certain embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22, 25, and 28.

On the other hand, the present application also provides nucleic acid molecules encoding the CAR described in the present application.

In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence as set forth in any one of SEQ ID 21, 24, and 27.

On the other hand, the present application also provides a vector, which contains the nucleic acid molecule described in the present application.

On the other hand, the present application also provides cells, which contain the CAR described in the present application, the nucleic acid molecules described in the present application, and/or the vectors described in the present application.

In certain embodiments, the cells comprise immune cells.

In certain embodiments, the cells are selected from the group consisting of: T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells.

In certain embodiments, the cells are T cells.

In certain embodiments, the T cells are autologous and/or allogeneic T cells.

In certain embodiments, the T cells are stem cell-derived autologous T cells and/or stem cell-derived allogeneic T cells.

In certain embodiments, the T cells are primary autologous T cells and/or primary allogeneic T cells.

In certain embodiments, the T cells comprise genetic modifications that include inhibiting or eliminating at least one endogenous gene expression, activity involved in responding to self- and/or allogeneic antigen recognition polypeptides.

In certain embodiments, the cell's endogenous TCR and MHC are not expressed or have low expression.

In certain embodiments, the cells include:

a) Low expression or no expression of endogenous HLA-I molecules,

b) Low or no expression of endogenous TCR molecules, and/or

c) Endogenous NKG2A molecules are poorly expressed or not expressed.

In some embodiments,

a) The low expression or no expression of endogenous HLA-I protein includes knocking out the gene encoding endogenous HLA-I,

b) The low expression or no expression of the endogenous TCR protein includes knocking out the gene encoding the endogenous TCR, and/or

c) The low expression or no expression of endogenous NKG2A molecules includes knocking out the gene encoding endogenous NKG2A.

In certain embodiments, gene knockout technology is used to knock out endogenous TRAC and/or B2M.

In certain embodiments, the gene knockout technology is CRISPR/Cas9 technology.

In certain embodiments, the cells include:

a) Use CRISPR/Cas9 technology to knock out endogenous B2M,

b) Use CRISPR/Cas9 technology to knock out endogenous B2M and TCR, or

c) Use CRISPR/Cas9 technology to knock out endogenous B2M, TCR, and NKG2A.

In certain embodiments, the gRNA used by the CRISPR/Cas9 technology includes a nucleotide sequence as shown in any one of SEQ ID NO: 36-38, or a combination thereof.

On the other hand, the present application also provides a pharmaceutical composition, which includes the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cells described in the present application, and a pharmaceutically acceptable accepted carrier.

On the other hand, the present application also provides a kit, which contains the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cells described in the present application, and/or the nucleic acid molecule described in the present application. The pharmaceutical composition.

On the other hand, the present application also provides methods for improving the survival time and/or expansion ability of tumor-targeted immune cells in the presence of host immune cells, including:

a) Provide immune cells;

b) Optionally, modifying the immune cell, the modification comprising inhibiting or eliminating at least one endogenous gene expression and activity involved in responding to autologous and/or allogeneic antigen recognition polypeptides;

c) modifying the immune cell with a polynucleotide encoding a CAR comprising an NKG2A antigen binding domain and a tumor antigen binding domain.

In certain embodiments, in step c), the method includes introducing the nucleic acid molecule described herein or the vector described herein into the immune cell.

In certain embodiments, the method further includes causing the immune cell to express a CAR described herein.

In certain embodiments, the method includes modifying the immune cell, the modification comprising inhibiting or eliminating at least one endogenous gene expression, activity involved in responding to a self- and/or allogeneic antigen recognition polypeptide.

In certain embodiments, the CAR includes:

a) NKG2A antigen-binding domain and tumor antigen-binding domain, CD28 or the transmembrane domain of CD8, the costimulatory signaling domain of CD28, and CD3ζ; and/or

b) NKG2A antigen-binding domain and tumor antigen-binding domain, the transmembrane domain of CD28 or CD8, the costimulatory signaling domain of 4-1BB, and CD3ζ; and/or

c) NKG2A antigen-binding domain and tumor antigen-binding domain, CD28 or the transmembrane domain of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and CD3ζ;

d) NKG2A antigen-binding domain and tumor antigen-binding domain, CD28 or the transmembrane domain of CD8, and CD3ζ.

In certain embodiments, the NKG2A antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 3-8; or a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a heavy chain variable region as set forth in SEQ ID NO: 1 The light chain variable region of the amino acid sequence shown in SEQ ID NO:2.

In certain embodiments, the tumor antigen is BCMA, and the BCMA antigen binding domain includes the amino acid sequence shown in SEQ ID NO:9-14; or the heavy chain including the amino acid sequence shown in SEQ ID NO:15 may variable region and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:16.

In certain embodiments, the antigen binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20, 23, and 26.

In certain embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22, 25, and 28. In certain embodiments, step b) includes:

i) Use CRISPR/Cas9 technology to knock out endogenous B2M,

ii) Use CRISPR/Cas9 technology to knock out endogenous B2M and TCR, or

iii) Use CRISPR/Cas9 technology to knock out endogenous B2M, TCR, and NKG2A.

In certain embodiments, the gRNA used by the CRISPR/Cas9 technology includes a nucleotide sequence as shown in any one of SEQ ID NO: 36-38, or a combination thereof.

In certain embodiments, the immune cells are selected from the group consisting of: T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells.

In certain embodiments, the immune cells are autologous or allogeneic T cells, stem cell-derived T cells, primary T cells, or autologous T cells derived from a human.

In certain embodiments, the methods are used to treat and/or prevent tumors.

In certain embodiments, the tumors include hematological tumors and/or solid tumors.

In certain embodiments, the hematological neoplasm comprises multiple myeloma.

In certain embodiments, the methods are performed under ex vivo conditions.

On the other hand, this application also provides the CAR described in this application, the nucleic acid molecules described in this application, the vectors described in this application, the cells described in this application, and the pharmaceutical compositions described in this application. For use in preventing, alleviating and/or treating tumors.

On the other hand, the present application also provides a method for preventing, alleviating and/or treating tumors, which includes administering the cells described in the present application and the pharmaceutical composition described in the present application to a subject in need.

On the other hand, this application also provides the CAR described in this application, the nucleic acid molecules described in this application, the vectors described in this application, the cells described in this application, and the pharmaceutical compositions described in this application, which are used for Prevent, mitigate and/or treat tumors.

In certain embodiments, the tumors include hematological tumors and/or solid tumors.

In certain embodiments, the hematological neoplasm comprises multiple myeloma.

It should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described below (such as embodiments) can be combined with each other to constitute a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows the killing effect of the tandem CAR-T cells in this application on NK cells in vitro.

Figure 2 shows the effect of the tandem CAR-T cells in this application on killing tumor cells in vitro.

Figure 3 shows the in vivo anti-tumor effect of tandem CAR-T cells in this application.

Figure 4 shows the cytokine secretion after co-incubation of tandem CAR-T cells and tumor cells in this application.

Figure 5 shows the resistance of tandem UCAR-T cells in this application to NK cell killing in vitro.

Figure 6 shows the cytokine secretion after co-incubation of tandem UCAR-T cells and tumor cells in this application.

Figure 7 shows the effect of tandem UCAR-T cells in the present application on inhibiting tumor growth in vivo.

Figure 8 shows the effect of the tandem UCAR-T cells in the present application on antagonizing NK cell killing and killing tumor cells in vitro.

Figure 9A shows the effect of tandem UCAR-T cells in the present application on inhibiting tumor growth in an orthotopic tumor model under NK cell conditions.

Figure 9B shows the effect of tandem UCAR-T cells in the present application on mouse survival in an orthotopic tumor model under NK cell conditions.

Figure 10 shows the specific infiltration of tumor tissue by tandem UCAR-T cells in this application.

Figure 11 shows that tandem UCAR-T cells in the present application do not cause graft-versus-host reactions.

Detailed ways

The present application provides a dual-antigen-specific chimeric antigen receptor (CAR) targeting BCMA antigen and NKG2A antigen. The CAR includes a BCMA antigen-binding domain and an NKG2A antigen-binding domain. The present application further provides nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions related to the CAR of the present application. The present application also provides methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal. T cells expressing the CAR described in this application have a strong ability to resist NK cells, thereby having better survival and/or proliferation capabilities and enhanced anti-tumor capabilities. In addition, by connecting anti-NKG2A and anti-tumor antigen CARs in series, the dual purposes of resisting host immune rejection (such as NK cell attack) and anti-tumor can be achieved using only one vector, which is conducive to simplifying process preparation, quality inspection, and quality control. process.

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the fields 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, where suitable methods and materials are 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, unless otherwise specified, the materials, methods, and examples of this application are illustrative only and not intended to be limiting. From this disclosure, those skilled in the art will appreciate that many changes or alterations can be made in the specific embodiments that are disclosed and still obtain the same or similar results without departing from the spirit and scope of this disclosure. This application is not limited in scope to the specific embodiments described herein, which are intended merely as illustrations of aspects of the application, and functionally equivalent methods and components are within the scope of the application. This application covers the subject matter of this application with variations and modifications for various uses and conditions.

Unless otherwise stated, the practice of this application will employ traditional techniques in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: 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); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, 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 this application, the description in range format is merely for convenience and brevity and should not be construed as an irreversible limitation on the scope of this application. Accordingly, descriptions of ranges should be considered to specifically disclose all possible subranges as well as individual values within that range.

The term "about" as used herein refers to the usual error range for each value that is readily known to those skilled in the art. References herein to "about" a value or parameter include references to the value or parameter itself. For example, descriptions of "about X" include descriptions of "X". In this article, "about" may mean an error range that is acceptable in the technical field;. For example, it may be meant that "about" a value or parameter within ±10% of a value or parameter, eg, about 5uM may include any number between 4.5uM and 5.5uM.

the term

The terms "comprises" or "includes" generally mean the inclusion of explicitly specified features, but not the exclusion of other elements.

In this application, the term "Chimeric Antigen Receptor" (Chimeric Antigen Receptor, CAR) generally refers to a fusion protein containing an extracellular domain capable of binding antigen and at least one intracellular domain. It may include an antigen (eg, tumor-associated antigen (TAA)) binding region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.

In this application, the term "antigen-binding domain" refers to a molecule that specifically binds to an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immune molecules, i.e., containing the antigen that specifically binds to the antigen ("immune response") Binding site molecule.

In this application, the term "antibody" generally refers to an immunoglobulin molecule or an immunologically active portion of an immune molecule, ie, a molecule that contains an antigen-binding site that specifically binds ("immunoreactively") an antigen. They can include intact antibody molecules (also called immunoglobulins) or fragments of antibody molecules that retain antigen-binding ability. In some cases, the term "antibody" is used interchangeably with the terms "immunoglobulin" and "antigen-binding domain." Antibodies generally include, but are not limited to, monoclonal antibodies, polyclonal antibodies, natural antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain antibodies Antibody molecules (e.g. scFv), single domain antibodies. In certain embodiments, an "antibody" may be a single chain polypeptide comprising a light chain or light chain variable region, a heavy chain or a heavy chain variable region. For example, it is a single-chain polypeptide composed of a light chain and a heavy chain; for example, it is a single-chain polypeptide composed of a light chain variable region and a heavy chain variable region; for example, it is a single-chain polypeptide composed of a light chain variable region and a heavy chain. A single-chain polypeptide; for example, a single-chain polypeptide composed of a light chain and a heavy chain variable region; for example, a polypeptide composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). For example, CH contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). CL consists of a structural domain. For example, VH and VL can be further divided into hypervariable regions, called complementarity-determining regions (CDRs), and interspersed more conserved regions, called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In this application, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds.

In this application, the term "bispecific" generally refers to having the property of recognizing and/or binding to two different sites. The site may be a certain number of amino acids or a peptide segment or a group of peptide segments thereof, such as a protein expressed on a cell surface. For example, the bispecific antigen-binding domain in this application may include two different targeting binding domains: the NKG2A antigen-binding domain and the tumor antigen-binding domain. Often "bispecific" is used interchangeably with "dual target." In some examples, "bispecific CAR" and "tandem CAR" can be used interchangeably, and both are CARs provided in this application.

In this application, the term "loop structure" usually refers to a curved, non-closed peptide segment, which can be a loop structure formed by an NKG2A antigen-binding domain or a tumor antigen-binding domain via a suitable connection method; for example, where The binding region H and binding region L of one binding domain are connected to form an antibody, and the binding region H (such as heavy chain or heavy chain variable region) and binding region L (such as light chain or light chain variable region) of the other binding domain are respectively Ligated to both ends of the antibody.

In this application, the term "binding region L" generally refers to a polypeptide comprising a functional fragment of a light chain (eg, light chain, light chain variable region). For example, the binding region L is a light chain, for example, the binding region L is a light chain variable region. For example, the first binding region L is the light chain of an antibody that recognizes NKG2A. For example, the first binding region L is the light chain variable region of an antibody that recognizes NKG2A. For example, the second binding region L is the light chain of an antibody that recognizes BCMA. For example, the second binding region L is the light chain variable region of an antibody that recognizes BCMA.

In this application, the term "binding region H" generally refers to a polypeptide comprising a functional fragment of a heavy chain (eg, heavy chain, heavy chain variable region). For example, the binding region H is a heavy chain, for example, the binding region H is a heavy chain variable region. For example, the first binding region H is the heavy chain of an antibody that recognizes NKG2A. For example, the first binding region H is the heavy chain variable region of an antibody that recognizes NKG2A. For example, the second binding region H is the heavy chain of an antibody that recognizes BCMA. For example, the second binding region H is the heavy chain variable region of an antibody that recognizes BCMA.

In this application, the term "C-terminus" generally refers to one of the two termini of a polypeptide chain, the amino acid residue at this terminus carrying a free α-carboxyl group (—COOH). In certain embodiments, the free alpha carboxyl group may also be amidated in certain peptide chains.

In this application, the term "N-terminus" generally refers to one of the two termini of a polypeptide chain, the amino acid residue at this terminus carrying a free alpha amino group (-NH2). In certain embodiments, the free alpha amino group may also be amidated or cyclized.

In this application, the term "light chain" generally refers to a smaller molecular weight peptide chain in an immunoglobulin, which may include a light chain variable region (VL) and a light chain constant region (CL). According to the difference in structure and constant region antigenicity, it can be divided into two categories: "kappa light chain" and "lambda light chain".

In this application, the term "heavy chain" generally refers to the larger molecular weight peptide chain in an immunoglobulin, which may include a heavy chain variable region (VL) and a heavy chain constant region (CL). According to the different antigenicity of their constant regions, they can be divided into five categories: μ, γ, α, δ and ε.

In this application, the term "light chain variable region" generally refers to the amino-terminal domain of an antibody light chain. The light chain variable region may be referred to as "VL." These domains are typically the most variable portion of the antibody light chain (relative to other antibodies of the same type) and may include complementarity determining regions (CDRs) or hypervariable regions (HVR) and framework regions (FRs).

In this application, the term "heavy chain variable region" generally refers to the amino-terminal domain of the antibody heavy chain. The heavy chain variable region may be referred to as "VH." These domains are typically the most variable portion of the antibody heavy chain (relative to other antibodies of the same type) and may include complementarity-determining regions (CDRs) or hypervariable regions (HVR) and framework regions (FRs).

In this application, the terms "linker" and "linker fragment" are used interchangeably and generally refer to an amino acid sequence with flexible characteristics. For example, the linker can be used to connect different functional modules in a chimeric polypeptide (or fusion protein, or chimeric protein), and its functions can include improving the folding and stability of the chimeric polypeptide. For example, the length of the linking peptide can vary widely, such as from about 1 to about 100, from about 3 to about 20, from about 5 to about 30, from about 5 to about 18, or from about 3 to about 8. A glycine/serine linker of amino acids. For example, the connecting peptide can be (GGGGS)n/(G4S)n, G is glycine, S is serine, and n is an integer greater than or equal to 1; for example, n can be 1, 2, 3, 4, or 5. Each binding region H or binding region L in this application can be connected through a suitable linker to achieve the purpose of this application.

In this application, the term "transmembrane domain" generally refers to the region of a protein sequence that spans the cell membrane. In certain embodiments, the region of the protein sequence that spans the cell membrane is typically an alpha-helical structure and contains mostly hydrophobic amino acids. In certain embodiments, the transmembrane domain can be obtained from a natural protein (eg, the transmembrane domain from CD8 or a functional derivative sequence thereof), or the transmembrane domain can be a synthetic, non-naturally occurring protein segment. , such as thermodynamically stable hydrophobic protein segments in cell membranes.

In this application, the term "costimulatory domain" generally refers to the intracellular domain of a costimulatory molecule that can provide an immune costimulatory signal, which is a cell surface molecule required for an effective lymphocyte response to an antigen. . The costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB.

In this application, the term "intracellular signaling domain" is also referred to as "primary signaling domain" and generally refers to a signaling sequence containing a so-called immunoreceptor tyrosine-based activation motif or ITAM. For example, primary signaling domains derived from CD3ζ, FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10 and DAP12. In certain embodiments, intracellular signaling domains transduce effector function signals and direct cells to perform specialized functions. Although the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that truncated portions of the intracellular signaling domain are used, such truncated portions may be used in place of the intact chain as long as they are capable of transducing effector function signals. Thus, primary intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce effector function signals. Intracellular signaling domain.

In this application, the term "hinge domain" generally refers to a stretch of amino acids between two domains of a protein that allows flexibility of the protein and/or movement of one or more domains relative to each other. For example, hinge domains derived from the IgG family (such as IgG1 and IgG4), IgD and other protein molecules, such as hinge domains from CD28 and HLA families.

The term "host-versus-graft reaction (HVGR)" generally refers to the development of a foreign graft as a result of the immunogenetic differences between the donor and the recipient (or host) during a foreign donor transplant. The donor will be recognized and attacked by immune cells (such as NK cells) in the host body, thereby inhibiting or eliminating the donor.

The term "graft-versus-host disease (GVHD)" generally refers to the recognition of normal host tissue by donor T lymphocytes due to the diversity of TCRs in exogenous transplanted donor T lymphocytes and incompatibility with host HLA molecules. The antigens on the host amplify and release a series of cytokines to attack host cells.

In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, such as a human patient. For example, the pharmaceutical composition described in the present application may comprise the nucleic acid molecule described in the present application, the vector described in the present application and/or the cells described in the present application, and optionally a pharmaceutically acceptable adjuvant.

In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include plasmids, liposomes, phages such as lambda phage or M13 phage, and viral vectors. A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.

In this application, the terms "tumor" and "cancer" generally refer to cells that exhibit at least partial loss of control of normal growth and/or development. For example, common tumors or cancer cells have often lost contact inhibition and may be invasive and/or have the ability to metastasize.

The term "nucleic acid molecule" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form, including any nucleotide sequence encoding a polypeptide of interest or a fragment thereof .

In this application, the term "endogenous" refers to nucleic acid molecules or polypeptides derived from the organism itself.

The term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell, or the expression level is insufficient to achieve the function when overexpressed; it covers any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous , heterologous and overexpressed nucleic acid molecules and peptides.

The terms "individual" and "subject" are interchangeable and include humans or animals from other species including, but not limited to, humans, mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.

The term "transplant immune rejection" means that after the host undergoes allogeneic tissue, organ, or cell transplantation, the foreign graft is recognized by the host's immune system as an "alien component" and initiates a response to the transplant. Immunological response of attack, destruction and clearance.

The terms "therapeutically effective amount," "therapeutically effective," "effective amount," or "in an effective amount" are used interchangeably herein to refer to a compound that is effective to achieve a specified biological outcome as described herein, The amount of a preparation, substance or composition, pharmaceutical composition, such as, but not limited to, an amount or dose sufficient to promote a T cell response. An effective amount of immune cells refers to but is not limited to: the number of immune cells that can increase, enhance or prolong anti-tumor activity; increase the number of anti-tumor immune cells or activated immune cells; promote IFN-γ secretion, tumor regression, Tumor shrinkage, tumor necrosis, and the number of immune cells.

The term "MHC" stands for histocompatibility complex, a collective term for all groups of genes encoding biocompatibility complex antigens. MHC, known as HLA antigens in human cells, plays an important role in the transplant response, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.

The term "Human leukocyte antigen" (HLA) is the gene encoding the human major histocompatibility complex and is closely related to the function of the human immune system. HLA includes class I, class II and class III gene parts. HLA class I is a heterodimer consisting of a heavy chain (α chain) and a light chain β2 microglobulin (B2M). HLA-II class genes include the HLA-D family, mainly HLA-DP, HLA-DQ and HLA-DR, etc., which are mainly distributed on the surface of professional antigen-presenting cells such as B lymphocytes, macrophages and dendritic cells.

Detailed description of the invention

The present application provides a chimeric antigen receptor (CAR). The CAR includes a bispecific antigen-binding domain. The bispecific antigen-binding domain includes an NKG2A antigen-binding domain and a tumor antigen-binding domain. The NKG2A antigen-binding domain The domain includes a first binding region L and a first binding region H, and the tumor antigen binding domain includes a second binding region L and a second binding region H.

antigen

"NKG2A antigen" or "NKG2A" usually refers to the NKG2A polypeptide, which is a member of the NKG2 transcript group. The heterodimeric inhibitory receptor CD94/NKG2A formed by NKG2A and CD94 is expressed on NK cells, αβ T cells, and γδ T cells. and on the surface of a subset of NKT cells. As used herein, "NKG2A" refers to any variant, derivative or isoform of the NKG2A gene or encoded protein. The NKG2A polypeptide has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% identical to the amino acid sequence encoded by the transcript expressed by the gene of NCBI GenBank Gene ID: 3821 %, at least about 98%, at least about 99% or 100% homology or identity to an amino acid sequence or fragment thereof, and/or may optionally include at most one or at most two or at most three conservative amino acid substitutions. In one example, the human NKG2A polypeptide has the sequence shown in SEQ ID NO: 42.

In one example, the NKG2A polypeptide is a human NKG2A polypeptide, including at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% with the amino acid sequence shown in SEQ ID No: 42 , an amino acid sequence or a fragment thereof that is at least about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include at most one or at most two or at most three conserved Amino acid substitutions.

"BCMA antigen" or "BCMA" usually refers to BCMA polypeptide, which is a B-cell maturation antigen and belongs to the TNF receptor superfamily. BCMA activates B cell proliferation and survival after binding to its ligand. BCMA is specifically highly expressed in plasma cells and multiple myeloma cells, but is not expressed in hematopoietic stem cells and other normal tissue cells. "BCMA" refers to any variant, derivative or isoform of the BCMA gene or encoded protein. The BCMA polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least Amino acid sequences or fragments thereof that are about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include at most one or at most two or at most three conservative amino acid substitutions . In one example, the human BCMA polypeptide has the sequence shown in SEQ ID NO: 43.

In one example, the BCMA polypeptide is a human BCMA polypeptide, including at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% with the amino acid sequence shown in SEQ ID No: 43 , an amino acid sequence or a fragment thereof that is at least about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include at most one or at most two or at most three conserved Amino acid substitutions.

Any tumor antigen may be used in the tumor-related embodiments described herein ("example" and "embodiment" are used interchangeably herein). Antigens are expressed as polypeptides or intact proteins or parts thereof. Tumor antigens in this application include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor α (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; casein Amidase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4 , EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe ); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside ( OAcGD2); Folate receptor; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integration B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR ; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of tumor necrotic zone; G protein-coupled receptor class C group 5-member D ( GPRC5D); Antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 ( GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6 -AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); Cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; Cyclin B1; V-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein casein amino acid kinase (LCK); A kinase-anchored protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA Fc fragment of the receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stroma Cellular antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 ( FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1).

In one example, a chimeric antigen receptor (CAR) recognizes a pathogen antigen, eg, for use in treating and/or preventing pathogen infection or other infectious disease, eg, in an immunocompromised subject. Pathogen antigens include but are not limited to: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens include but are not limited to: cytomegalovirus (CMV) antigen, Epstein-Barr virus (EBV) antigen, human immune Defective virus (HIV) antigen or influenza virus antigen.

In one example, the CAR of the present application specifically binds BCMA polypeptide and NKG2A polypeptide. In one example, the CAR binds to the extracellular domain of NKG2A polypeptides and BCMA polypeptides.

bispecific antigen binding domain

In the CAR provided by the present application, the tumor antigen binding domain (such as BCMA antibody), the heavy chain/heavy chain variable region (VH) and/or the light chain/light chain variable region of the tumor antigen binding domain (such as NKG2A antibody) (VH) can be positioned at any suitable location.

In this application, the NKG2A antigen-binding domain includes a first binding region L and a first binding region H, and the tumor antigen-binding domain includes a second binding region L and a second binding region H. The first binding region L and the third binding region A binding area H, the second binding area L, and the second binding area H are connected in the following manner:

a) From the N end to the C end: the first binding region L or the first binding region H - the first binding region H or the first binding region L - the second binding region H or the The second bonding area L—the second bonding area L or the second bonding area H,

b). From the N end to the C end: the second binding area H - the second binding area L - the first binding area L - the first binding area H, or

c) The first binding region L and the second binding region H are connected to form a first polypeptide, the second binding region L and the first binding region H are connected to form a second polypeptide, and the first A polypeptide is connected to the second polypeptide, wherein the first binding region L is not connected to the first binding region H, and the second binding region L is not connected to the second binding region H.

In one example, the first binding region L includes a first light chain or a first light chain variable region (VL).

In one example, the first binding region H includes a first heavy chain or a first heavy chain variable region (VH).

In one example, the second binding region L includes a second light chain or a second light chain variable region (VL).

In one example, the second binding region H includes a second heavy chain or a second heavy chain variable region (VH).

In one example, in the mode a, the first bonding area L is connected to the first bonding area H, and the second bonding area L is connected to the second bonding area H.

In one example, in the mode a, the first light chain is connected to the first heavy chain, and the second light chain is connected to the second heavy chain.

In one example, in the mode a, the first light chain variable region (VL) is connected to the first heavy chain variable region (VH), and the second light chain variable region (VL ) is linked to the second heavy chain variable region (VH).

In an example, in the mode a, the connections are as follows: NKG2A VL—NKG2A VH—BCMA VH—BCMAVL.

In an example, in the mode a, the connections are as follows: NKG2A VL—(G4S)3—NKG2A VH—(G4S)3—BCMA VH—(G4S)3—BCMA VL.

In an example, in the manner b, the connections are as follows: BCMA VH—BCMA VL—NKG2A VL—NKG2A VH.

In an example, in the mode b, the connections are as follows: BCMA VH—(G4S)3—BCMA VL—(G4S)3—NKG2A VL—(G4S)3—NKG2A VH.

In one example, in the mode c, the connections from the N end to the C end are as follows:

c1. The second bonding area H—the first bonding area L—the second bonding area H—the first bonding area L;

c2. The first bonding area H—the second bonding area L—the first bonding area H—the second bonding area L;

c3. The second bonding area L—the first bonding area H—the second bonding area H—the first bonding area L;

c4. The first bonding area L—the second bonding area H—the first bonding area H—the second bonding area L;

c5. The second binding area H—the first binding area L—the first binding area L—the second binding area H; or

c6. The first bonding area H—the second bonding area L—the second bonding area L—the first bonding area H.

In an example, in the mode c1, the connection is as follows:

The second VH - the first VL - the second VH - the first VL.

In an example, in the mode c2, the connection is as follows:

The first VH - the second VL - the first VH - the second VL.

In an example, in the mode c3, the connection is as follows:

The second VL - the first VH - the second VH - the first VL.

In an example, in the mode c4, the connection is as follows:

The first VL - the second VH - the first VH - the second VL.

In an example, in the mode c5, the connection is as follows:

The second VH - the first VL - the first VL - the second VH.

In an example, in the mode c6, the connection is as follows:

The first VH - the second VL - the second VL - the first VH.

In this application, the NKG2A antigen-binding domain includes a first binding region L and a first binding region H, and the tumor antigen-binding domain includes a second binding region L and a second binding region H. The first binding region L and the third binding region A binding region H, the second binding region L, and the second binding region H are connected to form a loop structure.

In one example, the loop structure is formed in the following manner: the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the binding region H and the binding region L of the antibody. Connect both ends.

In one example, the antibody includes: the binding region H and the binding region L in order from the N-terminus to the C-terminus.

In one example, the antibody includes in order from the N-terminus to the C-terminus: the binding region H, the binding region L; the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.

In one example, the binding region H connected to the antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the binding region L of the other antigen-binding domain is the first binding region L. Two binding regions L, the binding region H of the other antigen binding domain is the second binding region H.

In one example, the binding region H connected to the antibody is the second binding region H, and the binding region L connected to the antibody is the second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H.

In one example, the antibody includes in sequence from the N-terminus to the C-terminus: the binding region H and the binding region L; the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.

In one example, the binding region H connected to the antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the binding region L of the other antigen-binding domain is the first binding region L. Two binding regions L, the binding region H of the other antigen binding domain is the second binding region H.

In one example, the binding region H connected to the antibody is the second binding region H, and the binding region L connected to the antibody is the second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H.

In one example, the antibody includes: the binding region L and the binding region H in order from the N-terminus to the C-terminus.

In one example, the antibody includes in order from the N-terminus to the C-terminus: the binding region L, the binding region H; the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.

In one example, the antibody includes in order from the N-terminus to the C-terminus: the binding region L, the binding region H; the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.

In one example, the binding region H connected to the antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the binding region L of the other antigen-binding domain is the first binding region L. Two binding regions L, the binding region H of the other antigen binding domain is the second binding region H.

In one example, the binding region H connected to the antibody is the second binding region H, and the binding region L connected to the antibody is the second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H.

In a certain example, the first binding area L, the first binding area H, the second binding area L, and the second binding area H are connected in the following manner:

The second bonding area L—the first bonding area H—the first bonding area L—the second bonding area H.

In a certain example, the first binding area L, the first binding area H, the second binding area L, and the second binding area H are connected in the following manner:

The first bonding area L - the second bonding area H - the second bonding area L - the first bonding area H.

In one example, the first binding region L includes a first light chain or a first light chain variable region (VL).

In one example, the first binding region H includes a first heavy chain or a first heavy chain variable region (VH).

In one example, the second binding region L includes a second light chain or a second light chain variable region (VL).

In one example, the second binding region H includes a second heavy chain or a second heavy chain variable region (VH).

In one example, the first binding region L includes a first light chain, the first binding region H includes a first heavy chain, the second binding region L includes a second light chain, and the second binding region H contains the second heavy chain.

In one example, the first binding region L includes a first VL, the first binding region H includes a first VH, the second binding region L includes a second light chain, and the second binding region H includes Second heavy chain.

In one example, the first binding region L includes a first light chain, the first binding region H includes a first heavy chain, the second binding region L includes a second VL, and the second binding region H Contains second VH.

In one example, the first bonding area L includes a first VL, the first bonding area H includes a first VH, the second bonding area L includes a second VL, and the second bonding area H includes a first VL. 2VH.

In this application, the first VL is the VL of the NKG2A antibody, the first light chain is the light chain of the NKG2A antibody, the first VH is the VH of the NKG2A antibody, and the first The heavy chain is the heavy chain of the NKG2A antibody, the second VL is the VL of the antibody that recognizes the tumor antigen, the second light chain is the light chain of the antibody that recognizes the tumor antigen, and the second VH The second heavy chain is the VH of the antibody that recognizes the tumor antigen, and the second heavy chain is the heavy chain of the antibody that recognizes the tumor antigen.

In one example, the bispecific antigen-binding domain includes the following connection method from the N-terminus to the C-terminus:

BCMA VL—NKG2A VH—NKG2A VL—BCMA VH,

BCMA VH—NKG2A VH—NKG2A VL—BCMA VL,

NKG2A VL—BCMA VH—BCMA VL—NKG2A VH, or

NKG2A VH—BCMA VH—BCMA VL—NKG2A VL,

In one example, the bispecific antigen-binding domain includes the following connection method from the N-terminus to the C-terminus:

BCMA VL—NKG2A VL—NKG2A VH—BCMA VH,

NKG2A VL—BCMA VL—BCMA VH—NKG2A VH,

NKG2A VH—BCMA VL—BCMA VH—NKG2A VL, or

BCMA VH—NKG2A VL—NKG2A VH—BCMA VL,

In one example, each light chain/VL and heavy chain/VH in the CAR can be connected through a linker. The linker may comprise any suitable amino acid sequence for providing a portion of the CAR that is flexible in composition and length to the extracellular antigen-binding region.

In one example, the binding region H and the binding region L of one antigen-binding domain are connected through a linker to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are connected to both ends of the antibody through a linker respectively.

In one example, the linker includes Lin1 or (G4S)n, where n is an integer equal to or greater than 1, and the Lin1 includes the amino acid sequence shown in SEQ ID NO: 18. For example, n in (G4S)n is 1 or 3.

In one example, the binding region H and the binding region L of one antigen-binding domain are connected through Lin1 or (G4S)3 to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are connected to the antibody through a linker respectively. Connect both ends.

In one example, the binding region H and the binding region L of one antigen-binding domain are connected through Lin1 or (G4S)3 to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are connected to both sides of the antibody through G4S respectively. end connection.

In one example, the antibody includes the first binding region H and the first binding region L, and the N-terminus of the antibody is connected to the second binding region H or the second binding region L through G4S, and the antibody The N-terminal is connected to the second binding region L or the second binding region H through G4S.

In one example, the antibody includes the second binding region H and the second binding region L, and the N-terminus of the antibody is connected to the first binding region H or the first binding region L through G4S, and the antibody The N-terminal is connected to the first binding region L or the first binding region H through G4S.

In one example, the bispecific antigen-binding domain includes the following connection method from the N-terminus to the C-terminus:

BCMA VL—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VH;

BCMA VH—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VL;

NKG2A VL—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VH;

NKG2A VH—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VL;

BCMA VH—G4S—NKG2A VL—(G4S)3—NKG2A VH—G4S—BCMA VL;

BCMA VL—G4S—NKG2A VL—(G4S)3—NKG2A VH—G4S—BCMA VH;

NKG2A VL—G4S—BCMA VL—(G4S)3—BCMA VH—G4S—NKG2A VH; or

NKG2A VH—G4S—BCMA VL—(G4S)3—BCMA VH—G4S—NKG2A VL.

In one example, in the method a), the connection method from the N end to the C end includes the following:

BCMA VL—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VH;

BCMA VH—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VL;

NKG2A VL—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VH;

NKG2A VH—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VL;

BCMA VH—G4S—NKG2A VL—Lin1—NKG2A VH—G4S—BCMA VL;

BCMA VL—G4S—NKG2A VL—Lin1—NKG2A VH—G4S—BCMA VH;

NKG2A VL—G4S—BCMA VL—Lin1—BCMA VH—G4S—NKG2A VH; or

NKG2A VH—G4S—BCMA VL—Lin1—BCMA VH—G4S—NKG2A VL.

In one example, the CAR comprises the amino acid sequence described in any one of SEQ ID NO: 22, 25, and 28.

In one example, the second VL is the VL of the BCMA antibody, the second light chain is the light chain of the BCMA antibody, the second VH is the VH of the BCMA antibody, and the second heavy chain is the BCMA antibody. heavy chain.

In one example, each light chain/VL and heavy chain/VH of the antibody in the CAR can be connected through a linker. The linker may comprise any suitable amino acid sequence.

In one example, the linker includes Lin1 or (G4S)n, where n is an integer equal to or greater than 1; the Lin1 includes the amino acid sequence shown in SEQ ID NO: 18.

In one example, the linker is a glycine/serine linkage of about 1 to about 100, about 3 to about 20, about 5 to about 30, about 5 to about 18, or about 3 to about 8 amino acids in length. fragment, and consists of glycine and/or serine residues in the sequence.

In one example, the glycine/serine linker is a peptide of formula [GGGGS]n((G4S)n), where n is an integer from 1 to 10, 2 to 8, or 3 to 5. For example, n is 1, 2, 3 or 4.

In one example, NKG2A antibodies VL and VH can be connected through a linker fragment. In one example, NKG2A antibody VL and VH are connected by a linker fragment comprising a sequence shown in SEQ ID NO: 18, or by (G4S)3.

In one example, BCMA antibodies VL and VH can be linked through a linker fragment. In one example, the BCMA antibody includes the sequence shown in SEQ ID NO:9-14, also includes the sequence shown in SEQ ID NO:18 or (G4S)3; or includes the sequence shown in SEQ ID NO:15 and 16. The sequence shown also includes the sequence shown in SEQ ID NO:18 or (G4S)3; or includes the sequence shown in SEQ ID NO:39 or 40.

In one example, the NKG2A antibody includes VH, VL, and a linker fragment. In one example, the NKG2A antibody includes the sequence shown in SEQ ID NO:3-8, also includes the sequence shown in SEQ ID NO:18 or (G4S)3; or includes the sequence shown in SEQ ID NO:1 and 2. The sequence shown also includes the sequence shown in SEQ ID NO:18 or (G4S)3; or includes the sequence shown in SEQ ID NO:41.

In one example, the BCMA antibody and the NKG2A antibody are connected via a linker fragment. The linker fragment may comprise any suitable amino acid sequence. Preferably, the linker fragment connecting the BCMA antibody and the NKG2A antibody includes a glycine/serine linker, and the glycine/serine linker is a peptide of formula (G4S)n, where n is 1 to 10, 2 to 8 Or an integer from 3 to 5. Preferably, n is 1, 2, 3, 4. Preferably, the linker fragment connecting the BCMA antibody and the NKG2A antibody comprises a (G4S)3 sequence (comprising the BCMA antibody heavy chain and light chain and the NKG2A antibody heavy chain and light chain connected by the linker fragment, (G4S)3 is located in the BCMA antibody and between NKG2A antibodies).

In one example, the CAR includes (i) an NKG2A antibody light chain located near the amino terminus of the BCMA antibody heavy chain, with the linker segment between them, (ii) an NKG2A antibody heavy chain located near the amino terminus of the NKG2A antibody light chain , wherein the linking fragment is located between them, and (iii) a BCMA antibody light chain positioned near the amino terminus of the NKG2A antibody heavy chain, wherein the linking fragment is located between them; the linking fragment optionally includes, such as SEQ ID NO :Sequence shown in 18, G4S or (G4S)3. In one example, the CAR includes (i) a BCMA antibody light chain located near the amino terminus of the NKG2A antibody heavy chain, with the linker fragment located between them, (ii) a BCMA antibody heavy chain located near the amino terminus of the BCMA antibody light chain , wherein the linking fragment is located between them, and (iii) the NKG2A antibody light chain located near the amino terminus of the BCMA antibody heavy chain, wherein the linking fragment is located between them; the linking fragment optionally includes, such as SEQ ID NO: The sequence shown in 18, G4S or (G4S)3.

In one example, the BCMA antibody can be positioned adjacent to the amino terminus of the NKG2A antibody (with the linker fragment between them). In one embodiment, the NKG2A antibody is located near the amino terminus of the BCMA antibody (with the linker fragment between them).

In one example, CAR-T cells expressing the CAR provided by the application have a better in vitro killing effect on NK cells than CAR-T cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the application have a better in vitro killing effect on NK cells than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the present application have a better effect of inhibiting tumor growth in vivo than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the application have longer survival time and/or expansion ability than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the application have better anti-NK cell and tumor cell killing effects in an in vitro NK cell environment than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the application have better anti-NK cell and tumor growth-inhibitory effects in the in vivo NK cell environment than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided by the application have a better specific infiltration effect into tumor tissue than ucar-t cells that only target tumor antigens.

In one example, ucar-t cells expressing the CAR provided herein do not cause a graft-versus-host (GVHD) response.

In one example, the NKG2A antigen-binding domain includes an scFv of an antibody that recognizes an NKG2A polypeptide (also called an NKG2A antibody).

In one example, the NKG2A antigen-binding domain includes the light chain variable region (VL) and/or the heavy chain variable region (VH) of an NKG2A antibody. In one example, the NKG2A antigen-binding domain includes the light chain and/or heavy chain of an NKG2A antibody. In one example, the antibody heavy chain or VH of NKG2A includes one or more heavy chain CDRs (HCDR): HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, SEQ HCDR3 of the sequence shown in ID NO:5. In one example, the NKG2A antibody heavy chain or VH includes the sequence shown in SEQ ID NO: 3-5. In one example, the NKG2A antibody light chain or VL includes one or more light chain CDRs (LCDR): LCDR1 of the sequence shown in SEQ ID NO:6, LCDR2 of the sequence shown in SEQ ID NO:7, SEQ ID LCDR3 of the sequence shown in NO:8. In one example, the NKG2A antibody light chain or VL includes the sequence shown in SEQ ID NO: 6-8. In one example, the NKG2A antibody heavy chain or VH includes the sequence shown in SEQ ID NO: 1. In one example, the NKG2A antibody light chain or VL comprises the sequence set forth in SEQ ID NO:2. In one example, the NKG2A antibody or NKG2A antigen-binding domain includes a VH of the sequence shown in SEQ ID NO: 1 and/or a VL of the sequence shown in SEQ ID NO: 2. In one example, the NKG2A antibody or NKG2A antigen-binding domain includes the sequences shown in SEQ ID NO: 1 and 2.

In one example, the BCMA antigen-binding domain includes an scFv of an antibody that recognizes a BCMA polypeptide (also called a BCMA antibody). In one example, the BCMA antigen binding domain includes BCMA antibodies VL and/or VH. In one example, the BCMA antigen binding domain includes the light chain and/or heavy chain of a BCMA antibody. In one example, the BCMA antibody heavy chain or VH includes one or more heavy chain CDRs (HCDR): HCDR1 of the sequence shown in SEQ ID NO:9, HCDR2 of the sequence shown in SEQ ID NO:10, HCDR2 of the sequence shown in SEQ ID NO:10 HCDR3 of the sequence shown in NO:11. In one example, the BCMA antibody heavy chain or VH includes the sequence shown in SEQ ID NO: 9-11. In one example, the BCMA antibody light chain or VL includes one or more light chain CDRs (LCDR): LCDR1 of the sequence shown in SEQ ID NO:12, LCDR2 of the sequence shown in SEQ ID NO:13, SEQ ID LCDR3 of the sequence shown in NO:14. In one example, the BCMA antibody light chain or VL includes the sequence shown in SEQ ID NO: 12-14. In one example, the BCMA antibody heavy chain or VH comprises the sequence set forth in SEQ ID NO: 15. In one example, the BCMA antibody light chain or VL comprises the sequence set forth in SEQ ID NO: 16. In one example, the BCMA antibody or BCMA antigen-binding domain includes a VH of the sequence shown in SEQ ID NO: 15 and/or a VL of the sequence shown in SEQ ID NO: 16. In one example, the BCMA antibody or BCMA antigen-binding domain includes the sequences shown in SEQ ID NO: 16 and 17. In one example, the BCMA antibody or BCMA antigen-binding domain includes the sequence shown in SEQ ID NO: 46-50.

In one example, the CAR includes all CDR regions of the NKG2A antibody and the BCMA antibody. In one example, the CAR includes the sequence shown in SEQ ID NO: 3-14. In one example, the CAR includes the light chain variable region and the heavy chain variable region of an antibody that recognizes a NKG2A polypeptide and an antibody that recognizes a BCMA polypeptide. In one example, a CAR includes the sequences set forth in SEQ ID NO: 1, 2, 15, and 16.

In one aspect, the present application contemplates modification of the amino acid sequence of a starting antibody or fragment (eg, VH or VL) that results in a functionally equivalent molecule. For example, the VH or VL of the NKG2A antibody or BCMA antibody included in the CAR can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, such as the VH or VL of the NKG2A or BCMA antibody. 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% identity.

This application contemplates modification of the entire CAR molecule, for example, modification of one or more amino acid sequences of various domains of the CAR molecule, so as to produce a functionally equivalent molecule. The modifiable CAR molecule retains at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% of the starting CAR molecule , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % identity.

One of ordinary skill in the art will understand that the antibodies or antibody fragments of the present application can be further modified so that they vary in amino acid sequence (e.g., relative to wild type or the sequences provided herein) but have the desired activity. no change. For example, additional nucleotide substitutions can be made to the protein, resulting in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be replaced by another amino acid residue from the same side chain family. In another embodiment, the amino acid fragments may be replaced by amino acid fragments that are structurally similar but differ in sequence and/or composition from the side chain family members, e.g., conservative substitutions may be made in which the amino acid residues are replaced by amino acids with similar side chains. residues substituted.

leader sequence and hinge domain

The CAR provided herein may also include a leader sequence and/or a hinge domain. In one example, the antigen-binding domain is connected to the transmembrane domain directly or through a hinge. In one example, the hinge includes a CD8 hinge, for example, the CD8 hinge includes SEQ ID NO: 35 or a sequence that is 95-99% identical to SEQ ID NO: 35.

In one example, the antigen binding domain includes a leader sequence. In one example, the leader sequence can be positioned at the amino terminus of the BCMA antibody VH (eg, at the amino terminus of the BCMA antibody heavy chain). In one example, the leader sequence is located at the amino terminus of the BCMA antibody VL (eg, at the amino terminus of the BCMA antibody light chain). In one example, the leader sequence is located at the amino terminus of the NKG2A antibody VL (e.g., located at the amino terminus of the NKG2A antibody light chain). The leader sequence may include any suitable leader sequence. In one example, the leader sequence includes the amino acid sequence of SEQ ID NO:29. In one example, the CAR lacks a leader sequence.

transmembrane domain

The CAR provided herein may also include a transmembrane domain. The transmembrane domain can anchor the CAR to the cell membrane. The transmembrane domain of the CAR of the present application may include a transmembrane domain selected from the following proteins: the α, β, or ζ transmembrane domain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(CD11a, CD18), ICOS(CD278), 4-1BB(CD137), GITR , CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84 , CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8 ), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D and/or the transmembrane domain of NKG2C.

In one example, the CAR includes a CD8 transmembrane domain that has at least one, two or three modifications of the sequence set forth in SEQ ID NO:30, but no more than 20, 10 or 5 modifications, or is identical to SEQ ID NO:30. The amino acid sequence of NO:30 is a sequence with 95-99% identity. In one example, the CD8 transmembrane domain includes the sequence set forth in SEQ ID NO:30.

In one example, the CAR includes a CD28 transmembrane domain having at least one, two, or three modifications as set forth in SEQ ID NO:31, but no more than 20, 10, or 5 modifications, or the same as SEQ ID NO:31 The amino acid sequence shown in :31 is a sequence with 95-99% identity. In one example, the CD28 transmembrane domain includes the sequence set forth in SEQ ID NO:31.

intracellular signaling domain

The CAR provided by the present invention may also include an intracellular signaling domain. In one example, the intracellular signaling domain (also called a primary signaling domain) includes a signaling domain selected from the following protein molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ (FCER1G), FcRβ (FcεR1b), CD79a , CD79b, FcγRIIa.

In one example, the intracellular signaling domain includes the intracellular signaling domain of CD3ζ. The CD3ζ intracellular signaling domain may include at least 1, 2, or 3 modifications but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence shown in SEQ ID NO:34, or the same as SEQ ID NO:34 The amino acid sequences shown are sequences with 95-99% identity. In one example, the CD3ζ signal domain includes the amino acid sequence shown in SEQ ID NO:34.

In one example, the intracellular signaling domain of the CAR provided by the invention includes the human CD3ζ signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3ζ signaling domain and a CD28 costimulatory signaling domain.

costimulatory signaling domain

The CAR provided herein may also include a costimulatory signaling domain. In one example, the costimulatory signaling domain includes a protein functional signaling domain selected from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226),

SLAMF4(CD244,2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D.

In one example, the co-stimulatory signal domain 4-1BB includes at least 1, 2, or 3 modifications of the amino acid sequence shown in SEQ ID NO: 33, but no more than 20, 10, or 5 modified amino acid sequences, or an amino acid sequence with The amino acid sequence shown in SEQ ID NO:33 is a sequence with 95-99% identity. In one example, the costimulatory signal domain includes the sequence shown in SEQ ID NO:33.

In one example, the costimulatory signal domain CD28 includes at least 1, 2, or 3 modifications but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence shown in SEQ ID NO:32, or is identical to SEQ ID NO:32. The amino acid sequence shown in ID NO:32 is a sequence with 95-99% identity. In one example, the costimulatory signal domain includes the sequence shown in SEQ ID NO:32.

In one example, the intracellular signaling domain of the CAR provided by the invention includes the human CD3ζ signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3ζ signaling domain and a CD28 costimulatory signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3ζ signaling domain and a 4-1BB costimulatory signaling domain. In one example, the intracellular signaling domain of the CAR includes a CD3ζ signaling domain, CD28 and 4-1BB costimulatory signaling domains.

Exemplarily, the CAR of the present application includes the sequence shown in SEQ ID NO: 17, 22, 25 or 28.

Exemplarily, the CAR of the present application includes any one of the sequences shown in SEQ ID NO: 17, 22, 25 or 28 and any one of the sequences shown in SEQ ID NO: 19, 44 or 45. sequence.

cell

This application provides cells that include the CARs provided herein. The cells include immune cells derived from stem cells or lymphoid lineage. The CAR can activate the immune cells after binding to the target antigen; the CAR (also called tandem CAR) provided in this application includes a bispecific antigen-binding domain, and the bispecific antigen-binding domain includes the NKG2A antigen-binding domain and the tumor Antigen binding domain. The CAR provided by this application has been described above. The cells provided by this application include all its technical solutions.

In one example, cells of the present application include immune cells (eg, T cells, NKT cells) that recognize NKG2A polypeptides and tumor antigens. In one example, cells of the present application include immune cells that recognize NKG2A polypeptides and pathogen antigens.

In one example, in the presence of host immune cells (eg, NK cells), the immune cells of the present application have longer survival times and/or the ability to expand.

In one example, compared with immune cells including CARs that only recognize tumor antigens, the immune cells of the present application exhibit stronger cell killing effects on cells carrying target tumor antigens in vivo and in vitro.

The lymphoid lineage including B, T, and natural killer (NK) cells can provide antibody production, regulation of the cellular immune system, detection of exogenous reagents in the blood, detection of host exogenous cells, etc. Non-limiting examples of immune cells of the lymphoid lineage include T cells, natural killer T (NKT) cells, and their precursors, including embryonic stem cells and pluripotent stem cells (eg, stem cells that differentiate into lymphoid cells or pluripotent stem cells). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. T cells can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two effector Memory T cells: such as TEM cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, γδ T cells, or αβ T cells. Cytotoxic T cells (CTL or killer T cells) are T lymphocytes capable of inducing the death of infected somatic cells or tumor cells.

Immune cells (eg, T cells) can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from many sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

In one example, the subject's own immune cells can be engineered to express the CAR of the present application. In one example, immune cells from donors other than the subject (allogeneic) can be engineered to express the CAR of the present application. In one example, the immune cells are T cells. In one example, the T cells can be CD4+ T cells and/or CD8+ T cells. In one example, the immune cells are CD3+ T cells. In one example, the cells of the present application include a cell population collected from PBMC cells after stimulation with CD3 magnetic beads.

In certain aspects of the present application, T cells may be obtained from a blood sample collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll ^™ isolation technology. In a preferred aspect, cells from the individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and the cells placed in an appropriate buffer or culture medium for subsequent processing steps. Multiple rounds of selection may also be used in the context of this application. In some aspects, it may be necessary to perform selection procedures and use "unselected" cells during activation and expansion. "Unselected" cells can also be subjected to other rounds of selection.

The cells of the present application are capable of regulating the tumor microenvironment.

The source of unpurified CTL can be any source known in the art, such as bone marrow, fetal, neonatal or adult, or other hematopoietic cell sources, such as fetal liver, peripheral blood or umbilical cord blood. Various techniques can be used to isolate cells. For example, negative selection can initially remove non-CTL. mAbs are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages of positive and negative selection.

Most of the terminally differentiated cells can initially be removed by relatively crude dissociation. For example, magnetic bead separation can be used initially to remove large numbers of irrelevant cells. In certain embodiments, at least about 80%, typically at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.

Procedures for isolation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxicity conjugated to or in combination with mAbs agents, including but not limited to complement and cytotoxins; and using antibody panning attached to a solid matrix (eg, plate, chip, elutriation) or any other convenient technique.

Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying levels of sophistication, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels.

Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI). In certain embodiments, cells are collected in culture medium including 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, eg, sterile isotonic culture medium.

In one example, cells expressing tandem CAR also include immune cells with low or no expression of HLA class I molecules, TCR molecules, NKG2A molecules or combinations thereof. Low expression or no expression of TCR, B2M or NKG2A means that the expression of TCR, B2M or NKG2A in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% respectively. , at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. More specifically, low expression or no expression of TCR, B2M or NKG2A means that the content of TCR, B2M or NKG2A in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, respectively. %, 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 can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies for TCR, B2M or NKG2A.

Low or no expression of HLA class I molecules

In the transplantation of allogeneic cells (such as immune cells expressing tandem CAR), the deletion of HLA class I molecules in the allogeneic cells can reduce the host CD8+-mediated cellular immune rejection.

In one example, the present application provides immune cells that recognize tandem CARs of NKG2A polypeptides and tumor antigens and have low or no expression of endogenous B2M. In one example, the present application provides immune cells that recognize tandem CARs of NKG2A polypeptides and pathogen antigens and have low or no expression of endogenous B2M. In one example, immune cells expressing tandem CARs have longer survival times and/or the ability to expand in the presence of host immune cells (eg, NK cells). In one example, compared with immune cells expressing a CAR that only recognizes tumor antigens, immune cells expressing tandem CARs exhibit stronger cell killing effects in vitro and in vivo.

Low or no TCR expression

In one example, the present application provides immune cells that recognize NKG2A polypeptides and tumor antigens and have low or no expression of endogenous TCR; optionally, the immune cells have low or no expression of endogenous B2M.

In one example, the present application provides immune cells that recognize a tandem CAR of NKG2A polypeptide and tumor antigen and have low or no expression of endogenous TCR; optionally, the immune cells have low or no expression of endogenous B2M. In one example, the present application provides immune cells that recognize tandem CARs of NKG2A polypeptides and BCMA polypeptides and have low or no expression of endogenous TCR/B2M. In one example, in the presence of host immune cells (such as NK cells), the immune cells expressing the tandem CAR have a longer survival time and/or the ability to expand. In one example, compared with immune cells expressing a CAR that only recognizes tumor antigens, the above-mentioned immune cells expressing a tandem CAR exhibit stronger cell killing effects in vivo and in vitro.

Low or no expression of NKG2A

In one example, the present application provides immune cells that recognize a tandem CAR that recognizes NKG2A polypeptides and tumor antigens, and have low or no expression of endogenous NKG2A; optionally, the immune cells have low or no expression of endogenous B2M, Endogenous TCR has low or no expression, and endogenous B2M/TCR has low or no expression. In one example, the present application provides immune cells that recognize tandem CARs of NKG2A polypeptides and BCMA polypeptides and have low or no expression of endogenous TCR/B2M/NKG2A. In one example, in the presence of host immune cells (such as NK cells), the immune cells expressing the tandem CAR have a longer survival time and/or the ability to expand. In one example, compared with immune cells expressing a CAR that only recognizes tumor antigens, the above-mentioned immune cells expressing a tandem CAR exhibit stronger cell killing effects in vivo and in vitro.

gene knockout

Gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR, B2M or NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M. In one example, gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M. In one example, gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M/NKG2A.

Gene knockout technologies include Argonaute, CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology. Gene silencing technologies include but are not limited to: antisense RNA, RNA interference, microRNA-mediated translation inhibition, etc.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is used for genome editing. The system includes Cas, a protein that can modify DNA using crRNA as its guide, CRISPR RNA (crRNA), which contains the RNA that Cas uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA, usually in the form of a hairpin. ring form), forms an active complex with Cas), transactivating crRNA (tracrRNA, binds to crRNA, forms an active complex with Cas), and an optional fragment of the DNA repair template that directs the cellular repair process to allow the insertion of specific DNA sequence of DNA).

In one example, CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells. In one example, CRISPR/Cas9 usually uses plasmids or electroporation to deliver a complex containing nucleic acid fragments and recombinant proteins to target cells, such as gRNA and Cas9 ribonucleoprotein complex (RNP). The crRNA needs to be designed for each application because this is the sequence Cas9 uses to recognize and bind directly to target DNA in the cell. crRNA and tracrRNA can be combined together to form guide RNA (gRNA). The gRNA sequence of the present application can be represented by the gRNA targeting domain sequence. In one example, the gRNA sequence is a targeting DNA sequence. In one example, the gRNA sequence is a nucleic acid sequence that is completely or partially complementary to the gRNA targeting DNA sequence. In one example, the gRNA molecule includes a molecule in which the gRNA sequence and crRNA/TracrRNA form a complete Cas9 guide sequence.

In one example, methods provided herein include delivering to a cell one or more gRNA constructs and one or more Cas9 polypeptides or nucleic acid sequences encoding Cas9 polypeptides. In one example, one or more gRNA constructs, one or more Cas9 polypeptides are delivered via vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation or a nucleic acid sequence encoding a Cas9 polypeptide). In one example, crRNA and tracrRNA including the gRNA targeting domain are administered alone, or a complete RNA can be administered. CRISPR/Cas9 transgenes can be delivered via vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation. In this application, after using CRISPR/Cas9 technology to knock out the TCR/B2M gene of immune cells, they were sorted to obtain immune cells with low or no expression of TCR/B2M.

The application also provides nucleic acid molecules encoding one or more exogenous receptors (eg, CARs) described herein, and nucleic acid inhibitory molecules or gRNAs that target endogenous TCR, B2M, or NKG2A.

Exemplarily, the gRNA targeting NKG2A includes the sequence shown in SEQ ID NO: 38; the gRNA targeting TRAC includes the sequence shown in SEQ ID NO: 36; the gRNA targeting B2M includes the sequence shown in SEQ ID NO: 37 sequence shown.

In one example, tandem CARs encoding recognition of target antigens (exemplarily, NKG2A and BCMA tumor antigens) are introduced into T cells to generate immune cells provided herein, optionally targeting endogenous TCRs, B2M and /Or the nucleic acid inhibitory molecule of NKG2A or the nucleic acid molecule of gRNA is introduced into T cells. In one example, in vitro transcribed tandem CAR nucleic acid molecules, nucleic acid inhibitory molecules targeting endogenous TCR, B2M or NKG2A, or gRNA can be introduced into cells as a transient transfection. An exemplary artificial DNA sequence is one that includes portions of a gene joined together to form an open reading frame encoding a fusion protein. The DNA portions linked together can be from a single organism or from multiple organisms.

Nucleic acid molecules, vectors and their uses

In this application, the nucleic acid molecule may encode the chimeric polypeptide described in this application. For example, the nucleic acid molecule may be an isolated form of a nucleotide, a deoxyribonucleotide or a ribonucleotide of any length, or an analogue isolated from the natural environment or artificially synthesized, but may encode a nucleotide as described herein. Chimeric peptides. For example, the nucleic acid molecule may comprise the nucleic acid sequence shown in any one of SEQ ID NO: 21, 24 and 27.

Genetic modification of immune cells (eg, T cells or NKT cells) can be accomplished by transducing a substantially homogeneous population of cells with the nucleic acid molecule or a vector comprising the nucleic acid molecule. In one example, retroviral vectors (gamma-retrovirus or lentivirus) are used to introduce nucleic acid molecules into cells. For example, a polynucleotide encoding a tandem CAR can be cloned into a retroviral vector. Non-viral vectors can also be used. Transduction can use any suitable viral vector or non-viral delivery system. Tandem CARs can be constructed with accessory molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF- κB IRES, RUNX1IRES, p53IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, nonbaculovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers ( For example, 2A peptides, such as P2A, T2A, E2A and F2A peptides).

Other viral vectors that may be used include, for example, adenovirus, lentiviral and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus or herpesviruses, such as Epstein-Barr virus.

Non-viral methods can also be used to modify immune cells. For example, nucleic acid molecules can be introduced into immune cells by microinjection under lipofection, asialomucoid-polylysine conjugation, or surgical conditions. Other non-viral gene transfer methods include in vitro transfection using liposomes, calcium phosphate, DEAE dextran, electroporation and protoplast fusion. The nucleic acid molecules can also be first transferred into cell types that can be cultured in vitro (for example, autologous or allogeneic primary cells or their progeny), and then the cells (or their progeny) modified by the nucleic acid molecules can be injected into Inject into the subject's target tissue or systemically.

pharmaceutical composition

The present application also provides a pharmaceutical composition, which includes the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cells described in the present application, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present application may conveniently be provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixture.

Sterile injectable solutions can be prepared by incorporating the immune cells of the present application into the desired amount of an appropriate solvent and incorporating varying amounts of other ingredients as needed. Such compositions may be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, glucose, dextrose and the like. The composition can also be freeze-dried. The compositions may include auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening agents, preservatives, flavoring agents, pigments, and the like, This depends on the route of administration and formulation required.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Protection against the action of microorganisms can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical forms may be brought about by the use of agents which delay absorption such as aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune cells or their progenitors.

The compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride may be particularly useful in buffers containing sodium ions.

If desired, pharmaceutically acceptable thickening agents can be used to maintain the viscosity of the composition at a selected level. For example, methylcellulose is readily and economically available and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of thickening agent may depend on the agent chosen. It is important to use an amount that achieves the chosen viscosity. Obviously, the selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel or other liquid form, e.g. time-release form or liquid-filled form).

The number of cells in the composition to be administered will vary depending on the subject being treated. More effective cells can be administered in smaller quantities. The precise determination of the effective dose can be determined based on individual factors for each subject, including his or her size, age, sex, weight, and the subject's condition. Dosages can be readily determined by those skilled in the art from this application and knowledge in the art.

One skilled in the art can readily determine the amounts of cells and optional additives, vehicles and/or carriers in the composition and administered in the method. Typically, any additives (other than one or more active cells and/or one or more reagents) are present in phosphate buffered saline in an amount from 0.001% to 50% by weight solution, and the active ingredient is expressed in micrograms to Milligrams are present in the order, for example, from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, or from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt%. % or about 0.05 wt% to about 5 wt%. For any composition to be administered to animals or humans, the following results can be determined: toxicity, for example by determining the lethal dose (LD) and LD50 in a suitable animal model such as rodents such as mice; the dose of the composition, wherein The concentration of the ingredients and the time of application of the composition elicit the appropriate response.

Pharmaceutical compositions including the present application may be provided to a subject systemically or directly to induce and/or enhance an immune response to an antigen and/or to treat and/or prevent tumors, pathogenic infections, or infectious diseases. In one example, a composition of the invention is injected directly into the organ of interest (eg, an organ affected by a tumor). Alternatively, the compositions of the present invention are provided to the organ of interest indirectly, such as by administration into the circulatory system (eg, veins, tumor vasculature). Expansion and differentiation agents can be provided before, simultaneously with, or after administration of the composition to increase the production of T cells, NKT cells, or CTL cells in vitro or in vivo.

Cells of the present application may include purified cell populations. One skilled in the art can readily determine the percentage of immune cells of the invention in a population using a variety of well-known methods, such as fluorescence-activated cell sorting (FACS). In a population including the immune cells of the present application, suitable ranges for purity are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosage can be readily adjusted by one skilled in the art (e.g., reduced purity may require increased dosage). Cells can be introduced by injection, catheter, etc.

The composition of the present application may be a pharmaceutical composition including the immune cells or progenitor cells of the present application and a pharmaceutically acceptable carrier. Administration may be autologous or allogeneic. For example, immune cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived immune cells or their progeny (eg, from in vivo, ex vivo, or ex vivo sources) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the compositions of the present application, they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).

8. Purpose or method

This application provides the use of the CAR described in this application, the nucleic acid molecules described in this application, the vectors described in this application, the cells described in this application, and the pharmaceutical compositions described in this application in the preparation of medicines. The above drugs are used to prevent, alleviate and/or treat tumors.

The present application provides methods for preventing, alleviating and/or treating tumors, which include administering the cells described in the present application and the pharmaceutical compositions described in the present application to a subject in need.

The present application provides methods for inducing and/or increasing an immune response in a subject in need of a cell or pharmaceutical composition of the present application.

The cells or pharmaceutical compositions of the present application can be used to treat and/or prevent tumors in subjects. The cells or pharmaceutical compositions of the present application can be used to prolong the survival of subjects suffering from tumors. The cells or pharmaceutical compositions of the present application may also be used to treat and/or prevent pathogenic infections or other infectious diseases, such as in immunocompromised human subjects. Such methods include administering an effective amount of cells or pharmaceutical compositions of the present application to achieve the desired effect, whether alleviation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount may be provided in one or more administrations. Effective amounts can be provided in bolus doses or by continuous infusion.

In one example, cells or pharmaceutical compositions including the present application can be used to treat subjects with tumor cells that have low levels of surface antigen expression, such as due to relapse of disease, where the subject has received treatments that resulted in residual tumor cells. . In certain embodiments, tumor cells have a low density of target molecules on the tumor cell surface.

In one example, cells or pharmaceutical compositions including the present application can be used to treat a subject suffering from disease relapse, wherein the subject has received immune cells (e.g., T cells) that comprise a single administration of a CAR, the CAR comprising Intracellular signaling domains, including costimulatory signaling domains (eg, 4-1BBz CAR). In one example, tumor cells have a low density of tumor-specific antigens on the tumor cell surface. In one example, the disease is BCMA-positive tumors. In one example, the tumor cells have a low density of BCMA on the tumor cells. Such methods include administering an effective amount of cells or pharmaceutical compositions of the present application to achieve the desired effect, alleviate an existing condition or prevent recurrence.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result following treatment. The effective amount can be administered to the subject in one or more doses. For therapeutic purposes, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease or otherwise reduce the pathological consequences of a disease. Effective amounts are generally determined by the physician on a case-by-case basis and are within the ability of those skilled in the art. When determining the appropriate dosage to achieve an effective amount, several factors are generally considered. These factors include the age, sex and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the cells or pharmaceutical composition of the application administered.

For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of approximately ^{10 -10} are typically infused. After the immune cells of the present application are administered to a host and subsequently differentiated, T cells specific for a particular antigen are induced. The cells or pharmaceutical compositions of the present application may be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.

Non-limiting examples of tumors include hematological tumors (such as leukemias, lymphomas, and myeloma), solid tumors; solid tumors include: ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer Cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma and various cancers (including prostate cancer and small cell lung cancer ). Non-limiting examples of tumors include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendrogliomas, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), Chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphatic vessels Sarcoma, lymphatic endothelial sarcoma, liver cancer, cholangiocarcinoma, synovialoma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, adenocarcinoma, cystadenocarcinoma Carcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma tumors, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, leukemias, multiple myeloma, Waldenstrom's macroglobulinemia and severe chain diseases, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the cervix, epithelial carcinomas of the uterus and ovary, adenocarcinoma of the prostate, transitional squamous cell carcinoma of the bladder, B- and T-cell lymphomas (nodular and Diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the tumor is selected from the group consisting of blood cancers (e.g., leukemias, lymphomas, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer , prostate cancer, skin cancer, stomach cancer, glioblastoma and laryngeal cancer. In one example, the cells or pharmaceutical compositions of the present application can be used to treat and/or prevent solid tumors that are not suitable for conventional treatment measures or that relapse and are refractory, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, and thyroid cancer. , gastric cancer, colorectal cancer. In one example, the tumor is a hematological tumor.

Treatment goals herein may include alleviating or reversing disease progression and/or alleviating side effects, or treatment goals may include reducing or delaying the risk of relapse.

The present application provides methods for treating and/or preventing pathogenic infections (eg, viral, bacterial, fungal, parasitic, or protozoal infections), eg, in immunocompromised subjects. The method may include administering an effective amount of a cell or pharmaceutical composition of the present application to a subject suffering from a pathogenic infection. Exemplary viral infections that are susceptible to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.

The term "enhancement" refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein. For example, enhanced response may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% in responsiveness %, 75%, 80%, 85%, 90%, 95% or 98% or more increase. As used herein, "enhancing" may also refer to increasing the number of subjects that respond to a treatment, such as immune cell therapy. For example, an enhanced response may refer to the total percentage of subjects responding to treatment, where the percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.

In one example, cells expressing the CAR provided by the present application have longer survival time and/or expansion ability, specific infiltration into tumor tissue, and good resistance to NK cell attack and tumor suppression in a mouse subcutaneous tumor model. Growth effect, showing good function in treating solid tumors.

In one example, cells expressing the CAR provided by the present application have longer survival time and/or expansion ability in a mouse orthotopic blood tumor model, as well as good anti-NK cell attack and tumor growth inhibition effects, showing Good function in treating hematoma.

In one example, cells expressing the CAR provided herein target BCMA-positive tumors. In one example, cells expressing a CAR provided herein target multiple myeloma.

9. Kit

The present application provides a kit, which contains the CAR provided by the present application, the nucleic acid molecule provided by the present application, the vector provided by the present application, the cells provided by the present application, and/or the pharmaceutical composition provided by the present application. Kits provided herein are used to induce and/or enhance immune responses and/or treat and/or prevent tumors or pathogenic infections in subjects. In one example, a kit includes an effective amount of the cells of the present application and a pharmaceutical composition. In one example, the kit includes a sterile container; such container may be in the form of a box, ampoule, bottle, vial, tube, bag, sachet, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug. In one example, the kit includes a nucleic acid molecule encoding the CAR of the present application, which recognizes the antigen of interest in an expressible form, and can optionally be included in one or more vectors.

In one example, instructions are also included. Instructions typically include information regarding the use of the cells or pharmaceutical composition to treat and/or prevent tumors or pathogenic infections. In one example, the instructions include at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases, or symptoms thereof; precautions; warnings; indications; unindications ; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References. These instructions may be printed directly on the container, or as a label affixed to the container, or provided as a separate sheet, booklet, card or folder within or with the container.

This application includes, for example, Chinese patent application publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN10639 7593A, CN106467573A, CN104140974A, CN108884459A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A , CN109908176A, CN109880803A, CN110055275A, CN110123837A, CN110438082A, CN110468105A and, for example, international patent application publication numbers WO2017186121A1, WO2018006882A1, WO2015172339A8, WO20 18/018958A1, WO2014180306A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, 2018045811A1、WO2018108106A1、WO 2018 /219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/21 Those CAR-T cells disclosed in 9029 and their preparation Methods, antibodies.

The present application will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, 2002, or according to the manufacturer. Suggested conditions. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

The biological materials used in the examples of this application are shown in Table 1.

Table 1

name source T cells Primary PBMC isolated from healthy donors NK cells Primary PBMC isolated from healthy donors monocytes Primary PBMC isolated from healthy donors Cell line RPMI-8226 Purchased from ATCC MM.1S tumor cells Purchased from ATCC NPG immunodeficient mice Purchased from Beijing Weitongda Biotechnology Co., Ltd.

Example 1 Tandem CAR targeting BCMA and NKG2A and preparation of CAR-T cells.

Using conventional molecular biology methods in this field, BCMA and NKG2A were used as targets, and the vector PRRLsin was used to construct and express CAR3 polypeptide (SEQ ID NO: 22), CAR4 polypeptide (SEQ ID NO: 25), and CAR5 polypeptide (SEQ ID NO : 28), CAR-T3 cells, CAR-T4 cells, CAR-T5 cells, and BCMA-CAR-T cells of BCMA-CAR (SEQ ID NO: 17) polypeptide. The tandem fragments of the antigen-binding domain are shown in Table 2, and their amino acid sequences are shown in any one of SEQ ID NO: 20, 23 and 26 respectively.

Table 2.

fragment composition Concatenated fragments 3 BCMAVL-G4S-NKG2AVH-(G4S)3-NKG2AVL-G4S-BCMAVH Concatenated fragments 4 NKG2AVL-G4S-BCMAVH-(G4S)3-BCMAVL-G4S-NKG2AVH Concatenated fragments 5 NKG2AVL-G4S-BCMAVH-linker1-BCMAVL-G4S-NKG2AVH

Example 2 Tandem CAR-T kills NK cells in vitro

Take 5 × 10 ⁴ UTD cells (i.e., the T cell group that has not been transfected with viral vectors), as well as the BCMA-CAR-T, CAR-T3, CAR-T4, and CAR-T5 cells prepared in Example 1, and in vitro and The activated and expanded NK cells (aNK) were co-incubated for 18 hours according to the effective-target ratio E:T=1:3, 1:1, and 3:1, according to the kit instructions (CytoTox Non-Radioactive Cytotoxicity Assay, Promega Company), the supernatant was taken to measure lactate dehydrogenase (LDH) content, and the aNK cleavage efficiency in each group was calculated. As shown in Figure 1, tandem CAR-T cells in each group were able to kill NK cells.

Example 3 Tandem CAR-T kills tumor cells in vitro

The effector cells (UTD, BCMA-CAR-T, CAR-T3, CAR-T4, CAR-T5) and target cells (multiple myeloma cell line RPMI-8226) were divided into effector-target ratios of 1:3 and 1: 1 and 3:1 were incubated for a total of 18 hours, and tumor cell killing in vitro was detected. According to the kit instructions (CytoTox Non-Radioactive Cytotoxicity Assay, Promega Company), take the supernatant to measure lactate dehydrogenase (LDH) content, and calculate the target cell lysis efficiency of each group. The results are shown in Figure 2. Tandem CAR-T cells in each group were able to kill tumor cells.

Example 4 Anti-tumor effect of tandem CAR-T in vivo

5×10 ⁶ RPMI-8226 cells were subcutaneously inoculated into NPG immunodeficient mice (purchased from Beijing Vitongda Biotechnology Co., Ltd.) (the inoculation diary is D0). The average tumor ^volume was approximately 190 mm 11 days after inoculation, divided into 5 Groups: UTD cell group, BCMA-CAR-T cell group, CAR-T3 cell group, CAR-T4 cell group, CAR-T5 cell group, 5 cells in each group. 1 × 10 ⁶ cells of the above-mentioned UTD, BCMA-CAR-T, CAR-T3, CAR-T4, and CAR-T5 cells were injected into the tail vein respectively. After injection, body weight was measured twice a week (including group administration and the day of euthanasia). Use vernier calipers to measure and record the long and short diameters of the tumors, calculate the tumor volume, draw the tumor growth curve based on the tumor volume, and compare the differences in the tumor growth curves between each group (tumor volume: V = 1/2 × long diameter × short diameter ² ). As shown in Figure 3, each group of tandem CAR-T cells can inhibit tumor growth in vivo.

Example 5 Cytokine secretion by tandem CAR-T cells in vitro

Take UTD, BCMA-CAR-T, CAR-T3, and CAR-T5 cells, and use RPMI-8226 cells as target cells. T cells: target cells = 1:1 or T cells: target cells: autologous mononuclear cells. Cells were co-incubated at a ratio of 1:1:1. After 24 hours, the supernatant was taken for monitoring using cytokine bead detection technology (CBA) (as shown in Figure 4).

Example 6 Preparation of tandem UCAR-T cells

According to the reagent instructions (GeneArt ^TM Precision gRNA Synthesis Kit, Thermo Fisher), gRNA sequences targeting TCR, B2M, and NKG2A (SEQ ID NO: 36, 37, 38) were synthesized in vitro, using conventional CRISPR/Cas9 technology, that is, Cas 9 enzyme and gRNA was mixed at a ratio of 1:4 to form RNP, incubated at room temperature, mixed each group of cells with RNP, introduced the RNP complex into CAR-T cells using maxcyte electroporation instrument, and knocked out UTD, BCMA-CAR-T, CAR- The endogenous TRAC/B2M of T3, CAR-T4, and CAR-T5 cells was used to obtain UTD-DKO, BCMA-UCAR-T, UCAR-T3, UCAR-T4, and UCAR-T5 cells respectively, and the endogenous TRAC/B2M of CAR-T3 cells was deleted. TRAC/B2M/NKG2A yielded UCAR-T3-NKG2A KO cells. T cells that were not transfected with viruses and had endogenous TCR/B2M knockout were named UTD-DKO.

Example 7: Resistance function of tandem UCAR-T cells against NK cells in vitro

Take 5×10 ⁴ UTD-DKO, BCMA UCAR-T, UCAR-T3, UCAR-T4, and UCAR-T5 cells prepared in Example 6, and mix them with activated and expanded NK cells (aNK) in vitro at a ratio of 1:1 respectively. , 2:1 ratio for co-incubation, flow cytometry staining was performed at 0hr, 4hr, 24hr and 48hr to detect the ratio of UCAR-T cells and NK cells in the co-culture system. As shown in Figure 5, tandem UCAR-T can resist killing by NK cells.

Example 8. In vitro cytokine secretion of tandem UCAR-T cells

The in vitro cytokine secretion of tandem UCAR-T cells was detected according to the method described in Example 5, and the results are shown in Figure 6.

Example 9. In vivo anti-tumor function of tandem UCAR-T cells

5×10 ⁶ RPMI-8226 cells were subcutaneously inoculated into NPG mice. The average tumor volume was about 350mm ³ 12-14 days after inoculation. They were divided into 4 groups: UTD-DKO cell group, BCMA UCAR-T cell group, and UCAR-T3 cells. group, UCAR-T5 cell group, 5 animals in each group. 1.5×10 ⁶ UTD-DKO, BCMA UCAR-T, UCAR-T3 or UCAR-T5 cells were injected into the tail vein respectively. Tumor growth curves were drawn with reference to the method described in Example 4. The results are shown in Figure 7. Tandem UCAR-T cells in each group can inhibit tumor growth in vivo.

Example 10: In vitro anti-tumor activity and anti-NK activity of tandem UCAR-T cells in the presence of NK cells

Take 2 × 10 ⁵ RPMI-8226 cells that have been stained and labeled by CFSE, and inoculate them into a 96-well plate at a ratio of tumor cells: T cells: aNK cells = 1:2:1, culture for 5 days, and perform cell counting and Flow cytometry staining (Figure 8). Take 2×10 ⁵ CFSE-stained and labeled multiple myeloma cell lines MM.1S-GFP, according to tumor cells: T cells: aNK cells = 2:2:0, 2:2:1, 2:2 :2, inoculated into a 96-well plate and cultured for 5 days, followed by cell counting and flow cytometric staining (Figure 8). As shown in Figure 8, tandem UCAR-T cells have longer survival time and/or expansion ability, and can target and kill both tumor cells and NK cells.

Example 11. Anti-tumor activity of tandem UCAR-T cells in vivo in the presence of NK cells

2×10 ⁶ multiple myeloma cell line MM.1S-luciferase cells (MM.1S-luc) were inoculated into NPG mice through the tail vein. Nine days after inoculation, the tumor load was detected by fluorescence imaging at approximately 2-5×10 ⁴ Radiance. (p/s/cm2/sr), divided into 7 groups (UTD-DKO, BCMA UCAR-T, BCMA UCAR-T+NK-1, BCMA UCAR-T+NK-2, UCAR-T3-NKG2A KO and UCAR- T3-NKG2A KO+NK-1, UCAR-T3-NKG2A KO+NK-2), 5 animals in each group. On D10, 1 × 10 ⁶ NK cells (labeled as NK-1 as above) or 2 × 10 ⁶ NK cells (labeled as NK-2 as above) were injected into the tail vein, and NK cells were injected every two days thereafter. A total of 5 injections were used to simulate the presence of NK cells; after the first NK cell injection, 1×10 ⁶ UCAR-T cells were injected into the tail vein. After injection, fluorescence imaging was performed once a week to monitor the tumor burden. , measure body weight twice a week (including group administration and euthanasia day), draw tumor growth curves according to tumor burden, compare the differences in tumor growth curves between groups, and record the survival period of mice. The results are shown in Figure 9A-B. NK cells significantly weakened the anti-tumor activity of BCMA UCAR-T cells and reduced the survival period of mice in the BCMA UCAR-T cell group; while in the presence of low-dose and high-dose NK cells, Neither the anti-tumor activity of UCAR-T3 nor the survival of mice was significantly altered. It shows that tandem UCAR-T cells can resist NK cell killing and inhibit tumor growth in vivo.

Example 12. Specific infiltration of tandem UCAR-T cells into tumor tissue

UCAR-T cells were taken to treat RPMI-8226 subcutaneous tumor-bearing model mice (as described in Example 9), including tumor tissues of three groups of mice, UTD-DKO, BCMA UCAR-T, and UCAR-T3-NKG2A KO, as well as heart and liver. Spleen lung kidney normal tissue. After fixation with paraformaldehyde, sections were sectioned and immunohistochemical staining with anti-CD3e was performed. As shown in Figure 10, in the UCAR group, especially the tandem UCAR-T group, there was more T cell infiltration in the tumor tissue than in the BCMA UCAR-T group; no significant infiltration was detected in normal tissues (including heart, liver, spleen, lung, and kidney tissue). infiltration of T cells. This shows that tandem UCAR-T cells not only have longer survival time and expansion ability, but also specifically infiltrate into tumor tissue and are safe.

Example 13. Tandem UCAR-T cells do not cause graft-versus-host reaction

1×10 ⁷ UTD, BCMA UCAR-T, and UCAR-T3-NKG2A KO cells were injected into NPG mice respectively. The PBS group was used as a negative control. The body weight was measured twice a week, and the hair and basic conditions of the mice were observed.

As shown in Figure 11, mice in the UTD group began to experience significant weight loss from D25, significant hair loss and reduced activity from D30, and showed obvious symptoms of GVHD. The tandem UCAR-T group was similar to PBS. The mice grew normally and gradually gained weight. This indicates that UCAR-T3 cells do not cause a graft-versus-host (GVHD) response.

The embodiments described herein include this embodiment as any single embodiment or in combination with any other embodiments or portions thereof. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

sequence information

Claims (33)

Chimeric Antigen Receptor (CAR), characterized in that the CAR includes a bispecific antigen binding domain, the bispecific antigen binding domain includes an NKG2A antigen binding domain and a tumor antigen binding domain, and the NKG2A antigen binding domain includes The first binding region L, the first binding region H, the tumor antigen binding domain includes the second binding region L, the second binding region H, the first binding region L, the first binding region H, the third binding region The two binding regions L and the second binding region H are connected to form a loop structure. The CAR as claimed in claim 1, wherein the loop structure is formed in the following manner: the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody, and the binding region H, The binding region L is connected to both ends of the antibody respectively. The CAR as claimed in claim 2, wherein the antibody sequentially includes from the N-terminus to the C-terminus: The binding area H, the binding area L. The CAR of claim 3, wherein the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody. C terminal connection. The CAR of claim 4, wherein the binding region H connected to an antibody is the first binding region H, the binding region L connected to the antibody is the first binding region L, and the other The binding region L of the antigen-binding domain is the second binding region L, and the binding region H of the other antigen-binding domain is the second binding region H. The CAR of claim 4, wherein the binding region H connected to an antibody is a second binding region H, and the binding region L connected to an antibody is a second binding region L. The binding region L of the other antigen-binding domain is the first binding region L, and the binding region H of the other antigen-binding domain is the first binding region H. The CAR of claim 5 or 6, wherein the first binding region L includes a first light chain variable region (VL), and the first binding region H includes a first heavy chain variable region (VH), the second binding region L includes a second light chain variable region (VL), and the second binding region H includes a second heavy chain variable region (VH). The CAR of claim 7, wherein the first light chain variable region (VL) comprises the first light chain complementarity determining region (LCDR) as shown in any one of SEQ ID NO: 6-8. ) or a combination thereof. The CAR of claim 7 or 8, wherein the first heavy chain variable region (VH) comprises the first heavy chain complementarity determining region as shown in any one of SEQ ID NO: 3-5 (HCDR) or a combination thereof. The CAR according to any one of claims 7-9, wherein the first light chain variable region (VL) includes a first light chain complementarity determining region 1 (LCDR1), a first light chain complementarity determining region Region 2 (LCDR2), first light chain complementarity determining region 3 (LCDR3), the first LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, and the first LCDR2 includes the amino acid sequence shown in SEQ ID NO:7 The amino acid sequence of the first LCDR3 includes the amino acid sequence shown in SEQ ID NO:8. The CAR according to any one of claims 7 to 10, wherein the first heavy chain variable region (VH) includes the first heavy chain complementarity determining region 1 (HCDR1), the first heavy chain complementarity determining region 1 (HCDR1), the first heavy chain complementarity determining region 1 (HCDR1), Region 2 (HCDR2), first heavy chain complementarity determining region 3 (HCDR3), the first HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, and the first HCDR2 includes the amino acid sequence shown in SEQ ID NO:4 The amino acid sequence of the first HCDR3 includes the amino acid sequence shown in SEQ ID NO:5. The CAR according to any one of claims 1 to 11, wherein the tumor antigen is selected from CD19, GPC3, Claudin18.2, WT1, HER2, EGFR, BCMA or a combination thereof. The CAR according to any one of claims 7-12, wherein the second light chain variable region (VL) comprises the second light chain variable region (VL) as shown in any one of SEQ ID NO: 12-14. Chain complementarity determining region (LCDR) or combination thereof. The CAR of any one of claims 7-13, wherein the second heavy chain variable region (VH) comprises the second heavy chain variable region (VH) as shown in any one of SEQ ID NO:9-11. Chain complementarity determining region (HCDR) or a combination thereof. The CAR according to any one of claims 7-14, wherein the second light chain variable region (VL) includes a second light chain complementarity determining region 1 (LCDR1), a second light chain complementarity determining region Region 2 (LCDR2), second light chain complementarity determining region 3 (LCDR3), the second LCDR1 includes the amino acid sequence shown in SEQ ID NO:12, and the second LCDR2 includes the amino acid sequence shown in SEQ ID NO:13 The amino acid sequence of the second LCDR3 includes the amino acid sequence shown in SEQ ID NO: 14. The CAR according to any one of claims 7-15, wherein the second heavy chain variable region (VH) includes the second heavy chain complementarity determining region 1 (HCDR1), the second heavy chain complementarity determining region 1 (HCDR1), the second heavy chain complementarity determining region Region 2 (HCDR2), second heavy chain complementarity determining region 3 (HCDR3), the second HCDR1 includes the amino acid sequence shown in SEQ ID NO:9, and the second HCDR2 includes the amino acid sequence shown in SEQ ID NO:10 The amino acid sequence of the second HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11. The CAR of any one of claims 1-16, wherein the connection includes connection through a linker, the linker is selected from Lin1 or (G4S)n, wherein n is 1 or 3; The Lin1 includes the amino acid sequence shown in SEQ ID NO:18. A nucleic acid molecule encoding the CAR of any one of claims 1-18. A vector comprising the nucleic acid molecule of claim 19. A cell comprising the CAR of any one of claims 1-18, the nucleic acid molecule of claim 19, and/or the vector of claim 20. The cell of claim 20, wherein the cell is selected from the group consisting of T cells, NK cells, NKT cells, macrophages, CIK cells and stem cell-derived immune cells. The cell according to any one of claims 20-21, wherein the cell includes: a) Low expression or no expression of endogenous HLA-I molecules, b) Low or no expression of endogenous TCR molecules, and/or c) Endogenous NKG2A molecules are poorly expressed or not expressed. The cell of claim 22, wherein a) The low expression or non-expression of endogenous HLA-I protein includes knockout of the gene encoding endogenous HLA-I, b) The low expression or no expression of the endogenous TCR protein includes knockout of the gene encoding the endogenous TCR, and/or c) The low expression or non-expression of endogenous NKG2A molecules includes knockout of the gene encoding endogenous NKG2A. Pharmaceutical composition, comprising the CAR as described in any one of claims 1-17, the nucleic acid molecule as described in claim 18, the vector as described in claim 19, and/or any one of claims 20-23 The cells described in the item, and pharmaceutically acceptable carriers. A kit comprising the CAR as described in any one of claims 1-17, the nucleic acid molecule as described in claim 18, the vector as described in claim 19, and any one of claims 20-23 The cell, and/or the pharmaceutical composition of claim 24. A method for improving the survival time and/or expansion ability of tumor-targeted immune cells in the presence of host immune cells, comprising introducing into the immune cells a nucleic acid molecule as described in claim 18 or claim 19 the carrier. The method of claim 27, further comprising causing the immune cells to express the CAR of any one of claims 1-17. The method of any one of claims 26-27, further comprising modifying said immune cell, said modification comprising inhibiting or eliminating at least one endogenous gene involved in responding to autologous and/or allogeneic antigen recognition polypeptides expression, activity. The CAR of any one of claims 1-17, the nucleic acid molecule of claim 18, the vector of claim 19, the cell of any one of claims 20-23, the Use of the pharmaceutical composition described in 24 in the preparation of medicaments for preventing, alleviating and/or treating tumors. A method for preventing, alleviating and/or treating tumors, comprising administering the cell according to any one of claims 20 to 23 and the pharmaceutical composition according to claim 24 to a subject in need. The CAR of any one of claims 1-17, the nucleic acid molecule of claim 18, the vector of claim 19, the cell of any one of claims 20-23, claim 24 The pharmaceutical composition is used to prevent, alleviate and/or treat tumors. The use of claim 29, the method of claim 30, the CAR, nucleic acid molecule, vector, cell, pharmaceutical composition of claim 31, wherein the tumor includes a hematological tumor and/or a solid tumor. The use of claim 29, the method of claim 30, the CAR, nucleic acid molecule, vector, cell, pharmaceutical composition of claim 31, wherein the hematoma includes multiple myeloma.