Method for phase separation-based modification of chimeric antigen receptor, and use thereof

Abstract

A method for phase separation-based modification of a chimeric antigen receptor, and the use thereof. It has been discovered that the intracellular domain of CD28 can undergo phase separation with the intracellular domains of Lck and PD-1 and regulate T cell activation, and upon researching the molecular mechanisms of CD28 / Lck and CD28 / PD-1 phase separation, it has been discovered that two mutations of the intracellular domain of CD28 inhibits phase separation thereof with PD-1 and do not affect phase separation thereof with Lck. Incorporating the intracellular domain of CD28 having the two mutations in CAR-T design facilitates CAR-T resistance to PD-1 inhibition, and the therapeutic efficacy may be further improved by joint use with PD-1 blocking antibodies.

Description

A method for modifying chimeric antigen receptors based on phase separation and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to a prior application, patent application number 2023116043044, filed with the State Intellectual Property Office of China on November 28, 2023, entitled “A Method for Modifying Chimeric Antigen Receptors Based on Phase Separation and Its Application.” The entire text of this prior application is incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of tumor cell immunotherapy, and specifically relates to a new method for modifying chimeric antigen receptors based on phase separation, a recombinant gene designed based on the method, a vector and cells containing the gene, and applications thereof. Background Art

[0004] T cells are an important component of the body's adaptive immunity. Through the TCR (T Cell Receptor) on their membrane surface, they can specifically recognize MHC (Major Histocompatibility Complex) molecules presenting specific antigenic peptides on target cells, thereby initiating the activation pathway within the T cell and killing the target cell. In addition, the T cell activation process is also regulated by co-signaling receptors and cytokines. Among them, co-signaling receptors can be divided into co-stimulatory receptors (such as CD28, 4-1BB, etc.) and co-inhibitory receptors (such as PD-1, CTLA-4, etc.) based on their function.

[0005] The regulatory effect of co-signaling receptor molecules on T cell activation has been widely used in the clinical treatment of tumors and autoimmune diseases, and can be divided into immune blocking therapy and cell therapy. Among them, the combination therapy based on PD-1 and CTLA-4 antibodies has achieved good results in the clinical treatment of tumors, and the chimeric antigen receptor molecules (CAR) designed based on these receptor molecules (CD28 and 4-1BB) have also been widely used in T cell engineering and tumor treatment. At the same time, studies have found that better clinical effects can be achieved by combining immune blocking therapy and cell therapy. However, antibody blocking sometimes does not work well, which may be due to the ligand-independent signal of the receptor.

[0006] Published patents (CN201711457252, CN201780059525, and CN202111282362) involve combined therapies combining PD-1 blockade and CAR-T. Patent CN201880045582 involves the use of two CD28 mutants (mutations or deletions of YMNM and PRRP) in CAR-T or CAR-NK cancer treatment. Summary of the Invention

[0007] The present invention first provides a CD28 intracellular domain variant, wherein the variant comprises the following mutation sites:

[0008] CD28-modi3: R195S and R201S;

[0009] CD28-mRK:(185-188)SSSS:

[0010] CD28-mBRS1: (162-167)SSSSS;

[0011] CD28-mBRS2: (179-186)SSPGPTSS;

[0012] CD28-mPRS1: (178-183)SRRSGS;

[0013] CD28-mPRS2:(190-194)SYASS

[0014] CD28-mdBRS1:Δ(162-167);

[0015] CD28-mtPRS2:Δ(190-202);

[0016] or

[0017] CD28-modi1: G182P, T184K, D196Q, and A198T;

[0018] Or any combination of the above sites, preferably, comprising a combination of CD28-modi3 or CD28-mRK and the other sites mentioned above.

[0019] In a specific embodiment of the present invention, the variant has R195S and R201S mutation sites, or RKHY(185-188)SSSS mutation site, and optionally contains truncation, extension or conservative substitution.

[0020] In a specific embodiment of the present invention, the CD28 intracellular domain variant optionally further comprises:

[0021] 1) Arbitrary truncation or extension, based on the CD28 intracellular domain (162-202) sequence, the C or N-terminus is truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and tags or amino acids such as cysteine ​​are added to the C or N-terminus for labeling or separation. These variants are also known to those skilled in the art. When designing or preparing CAR molecules, these tags or amino acids can be removed.

[0022] 2) Any conservative mutation refers to the substitution of one amino acid by another amino acid within the same class, such as substitution of one acidic amino acid by another acidic amino acid, substitution of one basic amino acid by another basic amino acid, or substitution of one neutral amino acid by another neutral amino acid;

[0023] Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD28 intracellular domain (positions 162-202) sequence.

[0024] The present invention also provides a chimeric antigen receptor (CAR) that can specifically recognize a tumor-associated antigen (TAA) on cancer, wherein the CAR comprises the above-mentioned CD28 intracellular domain variant.

[0025] In another aspect, the present invention provides a chimeric antigen receptor (CAR) polypeptide comprising a TAA-binding region, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region, wherein the co-stimulatory signaling region comprises a region that increases CAR-T cell resistance to PD-1 inhibition.

[0026] In a specific embodiment of the present invention, the co-stimulatory signaling region comprises CD28 intracellular domain variant mRK and modi3.

[0027] In a specific embodiment of the present invention, the TAA-binding region is selected from a single-chain antibody against a tumor surface antigen, wherein the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab', a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor;

[0028] Preferably, the single-chain antibody is based on the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab.

[0029] In another specific embodiment of the present invention, the TAA-binding region in the CAR molecule also contains a protein domain based on natural ligand-receptor interaction, such as the extracellular domain of PD-1 protein (which can recognize PD-L1 / PD-L2 protein), the extracellular domain of CD2 (recognizing CD58 / CD59), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

[0030] In another specific embodiment of the present invention, the transmembrane domain is selected from the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor) or GITR; preferably, the transmembrane region of a type I single transmembrane molecule or a homologous multimer thereof; further preferably, it is the CD8 transmembrane domain.

[0031] The present invention also provides a nucleic acid sequence selected from:

[0032] i) encoding the above-mentioned CAR molecule; or ii) a nucleic acid sequence complementary to i).

[0033] The present invention also provides a nucleic acid construct, which contains the above-mentioned nucleic acid sequence;

[0034] Preferably, the nucleic acid construct is a vector;

[0035] More preferably, the nucleic acid construct is a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector, containing the above-mentioned nucleic acid sequence.

[0036] The present invention also provides a lentiviral vector system, which contains the above-mentioned nucleic acid sequence and lentiviral vector auxiliary components.

[0037] The present invention also provides a genetically modified cell, characterized in that the cell expresses the above-mentioned CAR molecule, or contains the above-mentioned nucleic acid sequence, or contains the above-mentioned nucleic acid construct, or is infected with the above-mentioned lentiviral vector system; preferably, the cell is selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and red blood cells, and the T cells include αβT cells, γδT cells, and regulatory T cells.

[0038] The present invention also provides a pharmaceutical composition or kit, which comprises the above-mentioned CAR molecule, the above-mentioned nucleic acid sequence, or the above-mentioned nucleic acid construct, or the above-mentioned lentiviral vector system or the above-mentioned gene-modified cell;

[0039] In a specific embodiment of the present invention, the pharmaceutical composition or kit comprises a checkpoint inhibitor;

[0040] In a specific embodiment of the present invention, the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

[0041] The present invention also provides the use of the above-mentioned CAR molecule, the above-mentioned nucleic acid sequence, or the above-mentioned nucleic acid construct, or the above-mentioned lentiviral vector system in preparing any one or more of the following use products: (1) preparing T cells or NK cells; (2) enhancing the proliferation ability of T cells or NK cells; (3) improving the killing ability of T cells or NK cells.

[0042] The present invention also provides the use of the above-mentioned CAR molecule, the above-mentioned nucleic acid sequence, or the above-mentioned nucleic acid construct, or the above-mentioned lentiviral vector system or the above-mentioned genetically modified cell in preparing any one or more of the following products: (1) treating cancer; (2) inhibiting the cytokine storm generated during cancer treatment;

[0043] Preferably, the cancer is selected from adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct carcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer and thyroid cancer, and / or optionally wherein the cancer is a blood cancer or a solid tumor cancer.

[0044] The present invention also provides a method for providing anti-tumor immunity in a subject suffering from a TAA-expressing cancer, the method comprising administering to the subject an effective amount of immune effector cells genetically modified to express the above-mentioned CAR polypeptide, thereby providing anti-tumor immunity in the subject.

[0045] In a specific embodiment of the present invention, the immune effector cells are selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells.

[0046] In a specific embodiment of the present invention, the method further comprises administering a checkpoint inhibitor to the subject.

[0047] In a specific embodiment of the present invention, the checkpoint inhibitor includes anti-PD-1 antibody, anti-PD-L1 antibody, PD-L1 recombinant protein and mutants thereof, PD-1 recombinant protein and mutants thereof, or a combination thereof.

[0048] Beneficial technical effects

[0049] In this study, we discovered that the intracellular domain of CD28 can phase separate from both Lck and PD-1 and regulate T cell activation. We investigated the molecular mechanisms of CD28 / Lck and CD28 / PD-1 phase separation and found that two mutations in the CD28 intracellular domain inhibit its phase separation from PD-1 but do not affect its phase separation from Lck. Introducing these two mutations in the CD28 intracellular domain into CAR-T design can help CAR-T cells resist PD-1 inhibition, and combined with PD-1 blocking agents can further enhance therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1: CD28 and Lck phase separate on a two-dimensional membrane (Figure 1A: The intracellular domain of the co-signaling receptor phase separates from the Lck truncation (top) and the full-length Lck (bottom) on a two-dimensional membrane; Figure 1B: Analysis results of the phase separation between the intracellular domain of the co-signaling receptor and the Lck truncation; Figure 1C: Analysis results of the phase separation between the intracellular domain of the co-signaling receptor and the full-length Lck).

[0051] Figure 2: Phase separation of CD28, CD38, and Lck in a solution system (Figure 2A: Colocalization of CD28 / pCD28 and CD3ε in phase-separated droplets; Figure 2B: Phase-separated droplets formed by CD28, CD3ε, and Lck exhibit excellent fluidity)

[0052] Figure 3: PD-1 regulates the phase separation of CD28 and Lck (Figure 3A: pPD-1 inhibits Lck UD-SH3Phase separation with pCD28, Figure 3B: pPD-1 inhibits the phase separation of LckUD-SH3-SH2 and pCD28, PD-1 does not affect Lck UD-SH3 and phase separation of LckUD-SH3-SH2 and CD28)

[0053] Figure 4: Doubly phosphorylated PD-1 mediates inhibition of pCD28 / Lck phase separation (Figure 4A: Doubly phosphorylated PD-1 intracellular domain inhibits two-dimensional phase separation of pCD28 and Lck; Figure 4B: Only phosphorylated PD-1 can be recruited to pCD28 / Lck phase-separated droplets; Figure 4C: Doubly phosphorylated PD-1 intracellular domain inhibits three-dimensional phase separation of pCD28 and Lck)

[0054] Figure 5: Recruitment of pPD-1 by phase-separated droplets of pCD28 / Lck (Figure 5A: PD-1 is phase-separated and excluded by pCD28 / Lck in a two-dimensional membrane system; Figure 5B: pPD-1 is phase-separated and recruited by pCD28 / Lck in a two-dimensional membrane system; Figure 5C: PD-1 is phase-separated and excluded by pCD28 / Lck in SLB-stimulated Jurkat cells; Figure 5D: pPD-1 is phase-separated and recruited by pCD28 / Lck in SLB-stimulated Jurkat cells)

[0055] Figure 6: Phase separation of PD-1 and CD28 is specific (Figure 6A: Two-dimensional phase separation of PD-1, costimulatory molecules, and CD3ε; Figure 6B: Two-dimensional phase separation of pPD-1, costimulatory molecules, and CD3ε)

[0056] Figure 7: CD28 mutants impair the phase separation of CD28 and PD-1 (Figure 7A: Confocal imaging of phase separation of CD28 and its mutants with Lck and PD-1, respectively; Figure 7B: Statistics of Lck aggregation mediated by CD28 and its mutants; Figure 7C: Statistics of PD-1 aggregation mediated by CD28 and its mutants)

[0057] Figure 8: CD28 mutants resist pPD-1 inhibition of their phase separation with Lck (Figure 8A: PD-1 regulates the phase separation of phosphorylated CD28 and its mutants with Lck; Figure 8B: Size statistics of phase-separated droplets)

[0058] Figure 9: CD28 mutants resist ligand-independent inhibition of PD-1 (Figure 9A: Three distributions of PD-1 at the Jurkat-Raji interaction surface; Figure 9B: Statistics of the three distributions of PD-1 at the interaction surface between CD28 and its mutant cells and Raji cells; Figure 9C: Residual activity statistics based on IL-2 secretion of CD28 and its mutant cells co-cultured with Raji cells expressing and not expressing PD-L1)

[0059] Figure 10: CD28 mutants enhance the ability of immune cells to resist PD-1 inhibition (Figure 10A: CD28 mutations enhance IL-2 secretion by CAR-T cells under PD-L1 inhibition; Figure 10B: Residual activity of CD28 and its mutant CAR-T cells under PD-L1 inhibition)

[0060] Figure 11: CD28 mutants enhance the effect of second-generation CAR-T combined with PD-L1 blockade

[0061] Figure 12: A: Human CD28 intracellular domain; B: CD28 variants of the present invention

[0062] Figure 13: Second-generation CAR-T therapy mouse experiment: bioimaging of mouse tumor burden (A) and survival curve (B). DETAILED DESCRIPTION

[0063] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0064] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0065] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0066] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0067] Technical terms

[0068] The CAR molecule provided by the present invention is defined by the following formula:

[0069] SP-TAA-HG-TM-CSR-ISD;

[0070] or

[0071] SP-TAA-HG-TM-ISD-CSR;

[0072] wherein "SP" represents an optional signal peptide,

[0073] Wherein "TAA" indicates tumor-associated antigen TAA-binding region,

[0074] wherein "HG" represents an optional hinge domain,

[0075] Wherein "TM" indicates transmembrane domain,

[0076] "CSR" indicates the costimulatory signaling region.

[0077] where "ISD" denotes intracellular signaling domain, and

[0078] Wherein “-” represents a peptide bond or a linker.

[0079] The signal peptide is located at the extracellular end of the CAR molecule. Its function is to guide the newly synthesized CAR protein into the endoplasmic reticulum of the cell. The CTR protein is glycosylated in the endoplasmic reticulum, and the glycosylated CTR can appear on the T cell membrane. The signal peptide sequence in any animal cell can be used on the CAR molecule. In the present invention, the signal peptide is optional and can be used or not.

[0080] Wherein, the tumor-associated antigen TAA-binding region recognizes a tumor surface antigen, and the TAA-binding region is preferably a single-chain antibody against a tumor surface antigen, wherein the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab', a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor.

[0081] Preferably, the single-chain antibody is derived from the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab.

[0082] Among them, the above-mentioned antigen recognition region also includes protein domains based on natural ligand-receptor interactions, such as the extracellular domain of PD-1 protein (which can recognize PD-L1 / PD-L2 protein), the extracellular domain of CD2 (recognizing CD58 / CD59), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

[0083] The "hinge region" herein refers to the hydrophilic region between the antigen recognition domain and the transmembrane domain. In the present invention, the hinge region is optional and may or may not be used. The hinge region can be derived from a variety of different antibodies or antigen receptors, particularly from CD4 molecules. In a specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, IgG4, and CD279 (PD-1).

[0084] Here, the "transmembrane region" only needs to include a peptide that can penetrate the cell membrane. The transmembrane region preferably used is the transmembrane region of a CD molecule. In one embodiment, the transmembrane region can be selected from, for example, the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor), or GITR. The transmembrane region of the present invention is preferably the transmembrane region of a type I single-pass transmembrane molecule or a homologous multimer thereof; in a preferred embodiment of the present invention, the CD8 transmembrane domain is used.

[0085] First generation CAR generally has the intracellular domain of CD3ζ chain, which is the main sender of the signal from endogenous TCR.Second generation CAR adds the intracellular signal transduction domain from various costimulatory protein receptors (such as CD28, 41BB, ICOS) to the intracellular domain of CAR, to provide other signals to T cells.Wherein, " costimulatory signal transduction region" refers to a part of CAR, which includes the intracellular domain of costimulatory molecules.Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens.The example of this molecule includes CD27, CD28 or its mutants, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and the ligand specifically bound to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3 and NKG2D. Therefore, although the CAR of the present invention mainly uses CD28 mutants as costimulatory signal elements, it can be used in combination with other costimulatory signal elements.

[0086] The preferred costimulatory signaling region of the present invention is a CD28 intracellular domain variant that increases the resistance of CAR-T cells to PD-1 inhibition.

[0087] The intracellular domain of CD28 in the present invention is positions 162-202 of the CD28 molecule (see FIG. 12A and SEQ ID NO: 1), and includes an optional expression or purification tag;

[0088] The mutation sites included in the CD28 variant are:

[0089] CD28-modi3: R195S and R201S (SEQ ID NO: 2);

[0090] CD28-mRK: (185-188)SSSS (SEQ ID NO: 3);

[0091] CD28-mBRS1: (162-167)SSSSS (SEQ ID NO: 4);

[0092] CD28-mBRS2: (179-186)SSPGPTSS (SEQ ID NO: 5);

[0093] CD28-mPRS1: (178-183)SRRSGS (SEQ ID NO: 6);

[0094] CD28-mPRS2: (190-194)SYASS (SEQ ID NO: 7);

[0095] CD28-mdBRS1: Δ(162-167) (SEQ ID NO: 8);

[0096] CD28-mtPRS2: Δ(190-202) (SEQ ID NO: 9);

[0097] or

[0098] CD28-modil: G182P, T184K, D196Q, and A198T (SEQ ID NO: 10);

[0099] or any combination of the above sites.

[0100] The above mutation sites are specifically shown in ( FIG12B ).

[0101] The above-mentioned CD28 intracellular domain variant optionally further comprises:

[0102] 1) Arbitrary truncation or extension, based on the CD28 intracellular domain (162-202) sequence, the C or N-terminus is truncated or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and tags or amino acids such as cysteine ​​are added to the C or N-terminus for labeling or separation. These variants are also known to those skilled in the art. When designing or preparing CAR molecules, these tags or amino acids can be removed.

[0103] 2) Any conservative mutation refers to the substitution of one amino acid by another amino acid within the same class, such as substitution of one acidic amino acid by another acidic amino acid, substitution of one basic amino acid by another basic amino acid, or substitution of one neutral amino acid by another neutral amino acid;

[0104] Optionally, the variant has greater than 95%, 96%, 97%, 98% or 99% sequence identity to the CD28 intracellular domain (positions 162-202) sequence.

[0105] Among them, the "intracellular signaling domain" is derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB) and FcεRIY (FCERIG).

[0106] In a specific embodiment, the intracellular signaling domain is derived from CD3ζ (TCRζ, GenBank accession number BAG36664.1). The T cell surface glycoprotein CD3zeta (CD3ζ) chain, also known as the T cell receptor T3zeta chain or CD247 (cluster of differentiation 247), is a protein encoded by the CD247 gene in humans.

[0107] As used herein, the terms "identity" or "homology" generally refer to the proportion of nucleotide bases or amino acid residues in a candidate sequence that are identical to those in a corresponding sequence, after alignment and, if necessary, introduction of gaps to achieve the maximum percent identity over the entire sequence, without considering any conservative substitutions as part of the sequence identity. N-terminal or C-terminal extensions or insertions should not be construed as reducing identity or homology. Methods and computer programs for alignment are available and well known in the art. For example, sequence identity can be determined using sequence analysis software.

[0108] The term "conservative substitution" refers to the substitution of one amino acid by another amino acid within the same class, for example, an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid. Exemplary substitutions are shown in the table below:

[0109] Nucleotide sequence and vector

[0110] The polynucleotide sequences of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0111] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0112] The nucleic acid construct provided by the present invention also includes one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence. The coding sequence of the CAR molecule of the present invention can be manipulated in a variety of ways to ensure the expression of the protein. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

[0113] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0114] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0115] The regulatory sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0116] Preferably, the nucleic acid construct is a vector.

[0117] Typically, the expression of the polynucleotide sequence encoding the CAR molecule is achieved by operably connecting the polynucleotide sequence encoding the CAR to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0118] The polynucleotide sequence encoding the CAR molecule of the present invention can be cloned into many types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Further, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector comprises a replication origin that works in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers.

[0119] More preferably, the nucleic acid construct is a lentiviral vector containing a replication origin, a 3'LTR, a 5'LTR and the aforementioned polynucleotide sequence.

[0120] The promoter can use a constitutive promoter sequence, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, the use of inducible promoters can also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably connected to an inducible promoter when the expression period is expressed, and turn off the expression when the expression is not expected. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

[0121] In order to evaluate the expression of the CAR molecule polypeptide or part thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that seeks to be transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a single DNA segment and used for a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences so as to be able to be expressed in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin, puromycin, and the like.

[0122] The reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of the regulatory sequence. After DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known technology to prepare or commercially obtain.

[0123] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0124] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0125] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0126] The present invention provides a lentiviral vector system comprising the aforementioned nucleic acid construct and lentiviral vector auxiliary components. The lentiviral auxiliary components include a lentiviral packaging plasmid and a cell line. The lentiviral vector system is constructed by viral packaging of the aforementioned nucleic acid construct with the assistance of the lentiviral packaging plasmid and cell line. The method for constructing the lentiviral vector system is commonly used in the art.

[0127] Cell therapy

[0128] The present invention also includes a type of cell therapy in which T cells are genetically modified to express the CAR molecules of the present invention (hereinafter referred to as CAR-T cells), and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells.

[0129] The CAR-T cells of the present invention include TRUCK, universal CAR, autonomous driving CAR, Armored CAR, self-destructive CAR, conditional CAR, labeled CAR, TanCAR, dual CAR or sCAR.

[0130] TRUCK (T cells redirected for universal cytokine-mediated killing) co-express a chimeric antigen receptor (CAR) and an anti-tumor cytokine. Cytokine expression can be constitutive or induced by T cell activation. Targeted specifically by the CAR, local production of pro-inflammatory cytokines recruits endogenous immune cells to the tumor site and may enhance the anti-tumor response.

[0131] Universal allogeneic CAR T cells are engineered to no longer express endogenous T-cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0132] Self-driving CAR T cells co-express CAR and chemokine receptors, which bind to tumor ligands and thus enhance tumor homing of CAR T cells.

[0133] CAR T cells engineered to resist immunosuppression (Armored CAR) can be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)) and can be used in conjunction with immune checkpoint switch receptors or with monoclonal antibodies that block immune checkpoint signaling.

[0134] Self-destructive CARs can be designed using RNA delivered by electroporation to encode the CAR. Alternatively, induction of apoptosis in T cells can be achieved based on ganciclovir binding to thymidine kinase in genetically modified lymphocytes or the more recently described system of activating human caspase 9 by small molecule dimers.

[0135] By default, conditional CAR T cells are unresponsive or "off" until a small molecule is added to complete the pathway, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells can be engineered to express receptors specific for a subsequent anti-tumor immune response elicited by the CAR-T cells, either active or passive. Alternatively, T cells can be engineered to express an adaptor-specific receptor with affinity for a subsequent secondary antibody directed against the target antigen.

[0136] The labeled CAR T cells express the CAR plus a tumor epitope to which an existing monoclonal antibody binds. Administration of the monoclonal antibody eliminates the CAR T cells and alleviates symptoms, with no other off-tumor effects, despite intolerable adverse reactions.

[0137] Tandem CAR (TanCAR) consists of two tandem single-chain variable fragments (scFv) fused with the intracellular domain of a T cell co-stimulatory molecule and the intracellular domain of CD3ζ. When the target cell expresses one of the two targets, TanCAR T cells can be activated.

[0138] Bi-CAR T cells express two independent CARs with different ligand-binding targets; one CAR contains only the intracellular domain of CD3ζ, and the other CAR contains only the intracellular domain of a co-stimulatory molecule. Activation of bi-CAR T cells requires simultaneous recognition of both targets co-expressed on the tumor.

[0139] The safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR are activated only when they encounter a target cell that has the standard CAR target but lacks the sCAR target.

[0140] Additionally, CAR-T-mediated immune responses can be part of an adoptive immunotherapy approach, in which CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR molecule.

[0141] Treatable cancers may be non-solid tumors, such as hematological tumors, eg, leukemias and lymphomas.

[0142] The present invention provides gene-modified T cells or pharmaceutical compositions containing the gene-modified T cells, wherein the cells contain the aforementioned polynucleotide sequence, or contain the aforementioned nucleic acid construct, or are infected with the aforementioned lentiviral vector system.

[0143] The CAR molecule modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include CTR cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0144] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0145] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient's disease signs and adjusting treatment accordingly.

[0146] Administration of the subject compositions can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0147] In some embodiments of the present invention, the CAR-T cells of the present invention or their compositions can be combined with other therapies known in the art. The therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, various radiotherapy preparations can be combined for treatment, including cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, and mycophenolic acid. In a further embodiment, the cell composition of the present invention is combined with bone marrow transplantation, T cell ablation therapy using chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (for example, before, simultaneously, or after) and administered to the patient.

[0148] In the present invention, "anti-tumor ability" refers to a biological effect, which can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0149] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0150] Optionally, the tumor is selected from one or more of leukemia and solid tumors.

[0151] Optionally, the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0152] Materials and methods

[0153] Antibody

[0154] The antibodies used in this invention were purchased from various commercial suppliers:

[0155] Biotin-labeled CD3ε antibody (UCHT1, Abcam, ab191112), FITC-labeled CD3ε antibody (OKT-3, Biolegend, 317306), PE-labeled Lck antibody (pY505, BD, 558552), PE-labeled Src antibody (pY418, BD, 560094), Csk antibody (Sino Biological, 200710-T44), Anti-Phospho-tyrosine (4G10, Millipore, 05-321), Goat anti-Rabbit IgG-APC (Solarbio, K0034G-APC), pCD3ζ antibody (pY142, Abcara, ab245764), CD3ζ antibody (Santa Cruz Biotechnology, sc_1239), pZap70 antibody (Cell Signaling Technology, 2701), Zap70 antibody (Abcam, ab32410), pPLCT1 antibody (Cell Signaling Technology, 14008), PLCT1 antibody (Santa Cruz Biotechnology, sc_7290), perk antibody (Cell Signaling Technology, 4370S), Erk antibody (Cell Signaling Technology, 4695S), CD3 antibody (eBioscience, 16-0037-85), APC-labeled CD3 antibody (eBioscience, 17-0038-42).

[0156] Chimeric molecule construction

[0157] All chimeric molecules are delivered using lentiviral vectors. From N-terminus to C-terminus, the entire chimeric molecule consists of an extracellular domain, hinge region, transmembrane domain, and intracellular domain. These four domains are sequentially linked by homologous recombination. The extracellular domain is a CD19 single-chain antibody (clone FMC63) sequence, using the CD8a signal peptide, hinge region, and transmembrane domain. The intracellular domain is a sequence of the intracellular domain of a costimulatory molecule (CD137, CD28, or a mutant thereof) linked to the intracellular domain of CD3ζ.

[0158] CAR cell construction based on primary human T cells

[0159] Using human CD3 + CD3-positive T cells were extracted from human PBMC using a T cell isolation kit (Biolegend, #480022) and then activated using human CD3 / CD28 Dynabeads (Thermo Fisher Scientific, #11161D). The cells were then infected with lentivirus loaded with CAR, and positive cells were sorted by flow cytometry and the surface expression of CAR was identified. NSG mice (6-8 weeks old, HFK Bioscience) were inoculated intravenously at 5.10 5 Nalm6-firefly luciferase-p2A-mCherry cells were recorded as day 0, and on the fourth day, various 1*10 6 CAR-T cell therapy was performed, and disease progression was monitored by bioluminescence imaging and survival analysis.

[0160] Jurkat cells

[0161] The chimeric molecules were transfected into Jurkat cells via lentiviral transfection for overexpression. Successfully transfected cells were sorted by flow cytometry and used for subsequent functional experiments. All Jurkat cells were cultured in RPMI1640 medium supplemented with 100 U / mL streptomycin, 100 μg / mL penicillin, and 10% fetal bovine serum at 37°C and 5% CO2.

[0162] Cell function experiments

[0163] 1×10 4The corresponding Jurkat cells were seeded in a U-shaped 96-well culture plate 30 minutes in advance, and the corresponding number of Raji cells expressing or not expressing PD-L1 were added. The total system was 100 μl and placed in a 37-degree Celsius incubator for 16-24 hours. In the PD-L1 blocking experiment, Raji cells expressing PD-L1 were incubated with PD-L1 blocking antibodies for 30 minutes, and then the corresponding number of second-generation CAR-Jurkat cells were added. The culture supernatant was collected and the secretion of IL-2 was detected by ELISA. Each experiment was repeated three times in parallel and three independent experiments were repeated.

[0164] Cell imaging experiments

[0165] 1×10 4 Jurkat cells were added to a 96-well optical imaging plate, and then an equal number of Raji cells expressing or not expressing PD-1 were added and co-cultured for 30 minutes. The cells were then imaged using a confocal microscope. PD-1 and CD28 on the Jurkat-Raji interaction surface were analyzed using Image J.

[0166] For two-dimensional cell imaging, biotin-containing SUVs were used as membranes. Streptavidin was first linked to the membrane, followed by biotin-labeled CD3ε single-chain antibody, CD28 antibody, and PD-L1. The corresponding Raji cells were added and incubated in an incubator for 30 minutes. The cells were then fixed with paraformaldehyde and stained with the appropriate immunofluorescence staining. Imaging and analysis were performed using TIRF-SIM.

[0167] Peptides

[0168] Unless otherwise specified, all peptides used in the present invention were synthesized by GL Biochemistry (Shanghai, China).

[0169] Recombinant protein expression and labeling

[0170] Lk

[0171] Full-length Lck, Lck regulatory domain constructs (various combinations of UD, SH3, and SH2 domains), or the Lck kinase domain were expressed in Expi293F cells, bacteria, and insect cells, respectively. To express full-length Lck, cDNAs encoding human wild-type and mutant Lck with an N-terminal 10× His tag (3-509a.a., Lck-wt; K273R & Y505F, Lck-open) were subcloned into the pHAGE vector. pHAGE-Lck-wt or pHAGE-Lck-open plasmids were transiently transfected into Expi293F cells using polyethyleneimine (PEI) (Polysciences, 23966). After 48 hours of expression, cells were harvested and lysed in 50 mM Na2HPO4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1X protease inhibitor cocktail, 1 mM PMSF, 0.3% Triton X-100, pH = 8.0. The lysate was centrifuged and filtered through a 0.22 μm membrane filter to obtain a clarified lysate containing the recombinant protein.

[0172] To express the Lck regulatory domain constructs, cDNAs encoding UD (3-60a.a.), UD-SH3 (3-121a.a.), UD-SH3-SH2 (3-226a.a.), or SH3-SH2 (59-226a.a.) with an N-terminal 10× His tag were subcloned into the pET28a vector and transduced into BL21(DE3) competent E. coli strains. Recombinant BL21 was induced with 0.25mM IPTG at 16°C for 12 hours for recombinant protein expression. Bacteria were harvested and lysed using an ultrasonic cell disruptor in 50mM Na2HPO4, 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM PMSF, pH=8.0. For UD recombinant proteins, the lysate was first boiled at 95°C for an additional 30 minutes. The clarified lysate containing the recombinant protein was then centrifuged through a 0.22μm membrane filter.

[0173] To express the Lck kinase domain (245-501a.a.), the encoding cDNA was inserted into the pFast Bac1-MBP vector, which contains an N-terminal MBP and HRV 3C sites and a 10×His sequence. Recombinant baculovirus containing the expression plasmid was generated using the Bac to Bac expression system. Baculovirus was produced in SF9 insect cells and used to infect BTI-Tn-5B1-4 insect cells for 48 hours. Cells were harvested and lysed using an ultrasonic cell disruptor in 50mM Na2HPO4, 300mM NaCl, 1mM EDTA, 1mM DTT, 1X protease inhibitor cocktail, 1mM PMSF, pH=8.0. The cleared lysate was incubated with dextrin beads (Smart-Lifesciences, SA026025). MBP was removed using 3C protease at 4°C for 12 hours. Cleaved recombinant Lck kinase was obtained after centrifugation.

[0174] PD-1 intracellular domain

[0175] The intracellular domain of PD-1 (193-288a.a.) and mutant cDNA sequences were inserted into the C-terminus of the pCold-GST plasmid and fused with GST and 8×His-tag for expression. The recombinant vector was transduced into the competent Escherichia coli BL21 (DE3) and cultured at 37°C. 0.5M IPTG was added and induced at 15°C for 24h. After collecting the bacteria, they were disrupted in PBS and the lysate supernatant was collected. The target protein was enriched using a GST affinity chromatography column and 3C enzyme was added for overnight enzyme digestion at 4°C. The flow-through was collected and then purified using Superdex75 molecular sieve. The target protein fraction was collected, concentrated, and then frozen at -80°C.

[0176] PD-L1 protein

[0177] The PD-L1 extracellular domain cDNA (19-238) was fused with a 10× His-tag and an AVI-tag and inserted into a pHAGE plasmid. The recombinant plasmid was transformed into Expi293F cells, and the culture supernatant was collected after 48 hours of expression. After concentration, the supernatant was purified using a His-tag purification column and a Superdex75 molecular sieve. The target protein fraction was collected and concentrated. The protein was biotinylated, desalted to remove excess biotin, and aliquoted for cryopreservation.

[0178] Fluorescent dye labeling

[0179] Proteins and peptides for fluorescent labeling were first reduced with TCEP supplement at a 10-fold protein molar ratio, followed by incubation with maleimides (C5-maleimide Alexa-488, C5-maleimide Alexa-546, and C2-maleimide Alexa-647, Thermo Scientific) at a 5-fold protein molar ratio and incubated at room temperature for 2 hours. Excess dye was removed by exchanging the buffer for PBS (Zeba spin desalting columns, Thermo Scientific).

[0180] Phase separation on loaded lipid bilayers

[0181] Small unilamellar vesicles (SUVs)

[0182] Phospholipids (95% DOPC, 5% DGS-NTA-Ni 2+ and 0.1% DSPE-PEG5000 or 95% POPC, 5% DSPE-PEG3350-Biotin) were dried in a 37°C water bath under a stream of nitrogen. The dried lipid film was further dehydrated in a desiccator for more than 2 hours and resuspended in PBS to a final concentration of 2 mg / ml. The lipid solution was sonicated for 5-10 minutes and then repeatedly frozen and thawed until the solution became clear. The solution was centrifuged at 21,380g for 60 minutes at 4°C. The supernatant containing SUVs was then collected.

[0183] Loaded lipid bilayer (SLB)

[0184] A glass-bottomed 96-well plate was washed with 5% Hellmanex III and rinsed thoroughly three times with MilliQ HO. The plates were then washed with 6M NaOH at 50°C for two hours, followed by a thorough rinse with MilliQ HO, and equilibrated with PBS for 30 minutes. 15 μl of freshly prepared SUVs were added and incubated at 37°C for one hour to induce SLB formation. The SLBs were then washed three times with aggregation buffer (PBS containing 1% BSA) and incubated at room temperature for 30 minutes.

[0185] On-membrane phase separation assay

[0186] Typically, 2 μM His-tagged wild-type or truncated Lck was incubated with preformed SLBs at room temperature for 1 hour. Unbound proteins were removed by washing three times with aggregation buffer. After an additional 30-minute incubation, 2 μM His-tagged CD3ε or 10 μM CD3 molecules (CD3ε, CD3δ, CD3γ, and CD3ζ without the His-tag) were added to the glass wells and mixed thoroughly to trigger cluster formation. The occupied area and Feret diameter of the formed clusters were measured using Image J.

[0187] Unless otherwise mentioned, all imaging experiments were performed on an Olympus FV1200 microscope equipped with a 60× oil immersion objective.

[0188] Phase separation determination in solution

[0189] Appropriate concentrations of CD3ε or PD-1 were premixed with CD28 and then added to Lck or its various truncated proteins, mixed rapidly. After incubation at room temperature for 1 hour, images were obtained using confocal microscopy, and cluster size was analyzed using ImageJ. For PD-1 recruitment experiments, CD28 and Lck were first formed into droplets, followed by the addition of PD-1 or its mutants. The mixture was incubated at room temperature for 30 minutes, and images were obtained using confocal microscopy. PD-1 recruitment was analyzed using ImageJ.

[0190] Example

[0191] Example 1 CD28 can phase separate from Lck on a two-dimensional membrane

[0192] Co-signaling receptors, as secondary signals for T cell activation, can influence T cell function by regulating TCR activation. Our previous research has shown that during TCR activation, the CD3ε subunit of the TCR complex can dissociate from the kinase Lck, thereby promoting TCR activation. However, the role of co-signaling receptors in this process remains unclear.

[0193] We used an in vitro reconstituted two-dimensional membrane system linked to the kinase Lck molecule to test several important co-signaling receptors. We found that only CD28 could phase separate with Lck on the two-dimensional membrane (Figure 1). Furthermore, CD28 was well recruited to phase-separated droplets of CD3ε / Lck. In solution, phase-separated droplets formed by CD28, CD3ε, and Lck also fused well (Figure 2).

[0194] Example 2 Phase-separated droplets recruit pPD-1 (phosphorylated PD-1)

[0195] Studies have shown that CD28 is the primary target of PD-1-mediated T cell inhibition, so we tested the effect of PD-1 on CD28 / Lck phase separation. Results showed that PD-1 did not significantly affect the phase separation of CD28 or pCD28 (phospho-CD28) from Lck. However, pPD-1 (phospho-PD-1) mediated the dissolution of pCD28 / Lck phase-separated droplets (Figure 3). The intracellular domain of PD-1 contains two phosphorylation sites: ITIM (immunotyrosine-based inhibitory motif) and ITSM (immunotyrosine-based switch motif). We tested monophosphorylated PD-1 at both sites and found that monophosphorylation of PD-1 facilitated its recruitment to pCD28 / Lck phase-separated droplets, while only doubly phosphorylated PD-1 directly mediated the inhibition of pCD28 / Lck phase separation (Figure 4). At the same time, we analyzed the distribution of PD-1 and pPD-1 on reconstituted phospholipid membranes and inside cells, respectively. The results showed that PD-1 was excluded from the phase-separated droplets of pCD28 / Lck, while pPD-1 was recruited (Figure 5).

[0196] Example 3 PD-1 can directly separate from CD28

[0197] Further mechanistic studies demonstrated that PD-1 can directly phase separate from CD28, and that pPD-1 mediates the dissolution of pCD28 / Lck phase-separated droplets by competitively binding to pCD28. Moreover, the phase separation of PD-1 and CD28 is specific and does not phase separate from other co-stimulatory receptor molecules (Figure 6).

[0198] Example 4 mRK and modi3 CD28 variants do not affect the phase separation of CD28 and Lck, but greatly weaken the phase separation of CD28 and PD-1

[0199] Through molecular dynamics simulations, we investigated the molecular mechanism of phase separation between PD-1 and CD28. The results showed that PD-1 primarily binds to the C-terminal portion of CD28, while Lck primarily binds to the N-terminal BRS region of CD28. Based on this, we designed two mutants (mRK and modi3) targeting the C-terminal region of CD28 and characterized their phase separation with PD-1 and Lck on a two-dimensional membrane. The results showed that the two mutations did not affect the phase separation of CD28 and Lck, but significantly weakened the phase separation of CD28 and PD-1 (Figure 7).

[0200] Example 5: Engineering Chimeric Antigen Receptors Based on Phase Separation

[0201] Immunotherapy based on PD-1 blockade has been widely used in cancer and other related diseases, but some patients still do not respond. We speculate that this may be due to the ligand-independent activity of PD-1. Studies have shown that PD-1 is phosphorylated at low levels even in the absence of PD-L1. Our co-culture of cells also revealed that even when target cells do not express PD-L1, some PD-1 is still recruited to cell-cell interfaces. Compared with wild-type CD28, CD28-mRK and CD28-modi3 inhibit this nonspecific recruitment. We also found that the two mutant CD28s can effectively resist the inhibition of pPD-1 on its dissociation from Lck (Figure 8). Functional experiments based on IL-2 secretion also demonstrated that CD28-mRK and CD28-modi3 can better resist the ligand-independent inhibitory effects of PD-1 (Figure 9).

[0202] Next, we introduced these two mutations into the design of a second-generation CAR-T (28z: CD28 intracellular domain + CD3ζ intracellular domain) and transferred them into Jurkat cells expressing PD-1 and those not expressing PD-1, respectively. The cells were then stimulated by co-culturing with Raji-B cells expressing PD-L1. The results showed that the mutations did not affect IL-2 secretion by T cells not expressing PD-1, but increased IL-2 secretion by T cells expressing PD-1. Analysis of the residual activity of T cells under PD-1 inhibition revealed that CAR-T containing these two CD28 mutants had higher residual activity (Figure 10). These results suggest that introducing CD28 mutations into CAR-T design significantly improves its ability to resist PD-1 inhibition.

[0203] We further tested the effect of CAR-T combined with PD-L1 blockade. The corresponding number of Raji cells expressing PD-L1 were pre-incubated with PD-L1 blocking antibodies in an incubator for 30 minutes, and then the corresponding Jurkat cells expressing PD-1 that had been transferred into the second-generation CAR were added and cultured together. By detecting IL-2 in the culture supernatant, we found that CAR-T cells containing CD28-mRK and CD28-modi3 can secrete more IL-2, which is similar to the effect of CAR-T cells containing 4-1BB (CD137) (Figure 11). This shows that by introducing CD28 mutations in the design of second-generation CAR-T, the PD-1 non-ligand-dependent inhibitory activity that occurs in the combined treatment of CAR-T and PD-1 antibodies can be compensated.

[0204] Example 6

[0205] In order to study the therapeutic effect of the second-generation CAR-T with CD28 mutation in vivo, we prepared CAR-T cells using human primary T cells and treated NSG mice inoculated with NALM6-based tumor cells. The results showed that compared with the PBS control group, the original second-generation CAR-T showed good therapeutic effects, and the therapeutic effect of CAR-T containing 4-1BB was slightly stronger than that of CAR-T containing CD28 (Figure 13). The introduction of CD28 mutations significantly improved its therapeutic effect, and CAR-T cells containing CD28-mRK achieved a therapeutic effect comparable to that of 4-1BB CAR-T (Figure 13).

Claims

1. A CD28 intracellular domain variant, wherein the mutant comprises the following mutation sites: CD28-modi3: R195S and R201S; CD28-mRK: (185-188)SSSS; CD28-mBRS1: (162-167)SSSSS; CD28-mBRS2: (179-186)SSPGPTSS; CD28-mPRS1: (178-183)SRRSGS; CD28-mPRS2:(190-194)SYASS CD28-mdBRS1:Δ(162-167); CD28-mtPRS2:Δ(190-202); or CD28-modi1: G182P, T184K, D196Q, and A198T; Or any combination of the above-mentioned sites; preferably, comprising a combination of CD28-modi3 or CD28-mRK and the above-mentioned other sites.

2. The CD28 intracellular domain variant of claim 1, further comprising: 1) Any truncation or extension, C or N-terminal truncation or extension of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids based on the CD28 intracellular domain (162-202) sequence; 2) Any conservative mutation, which means that one amino acid is replaced by another amino acid in the same category, such as one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another neutral amino acid; Optionally, the variant has a sequence identity of greater than 95%, 96%, 97%, 98% or 99% with the CD28 intracellular domain (162-202) sequence.

3. The CD28 intracellular domain variant according to claim 1 or 2, wherein the variant has R195S and R201S mutation sites, or RKHY(185-188)SSSS mutation site, and optionally comprises truncation, extension or conservative substitution.

4. A chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises the CD28 intracellular domain variant according to any one of claims 1 to 3.

5. A chimeric antigen receptor (CAR) polypeptide comprising a tumor-associated antigen (TAA)-binding region, a transmembrane domain, an intracellular signaling domain and a co-stimulatory signaling region, wherein the co-stimulatory signaling region comprises a region that increases CAR-T cell resistance to PD-1 inhibition; preferably, the co-stimulatory signaling region comprises a CD28 intracellular domain variant as described in any one of claims 1-3.

6. A chimeric antigen receptor (CAR) polypeptide as claimed in claim 5, wherein the TAA-binding region is selected from a single-chain antibody against a tumor surface antigen, wherein the tumor surface antigen is selected from one or more of CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein, Glypican-3, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, claudin18.2 and GD2; the single-chain antibody is selected from a single-chain antibody fragment, a single-chain Fv (scFv), a single-chain Fab, a single-chain Fab′, a single-domain antibody fragment, a single-domain multispecific antibody, an intracellular antibody, a nanobody or a single-chain immune factor; Preferably, the single-chain antibody is based on the following monoclonal antibodies: Inebilizumab, Rituximab, Ofatumumab, Glofitamab, Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab; The TAA-binding region in the CAR molecule also contains protein domains based on natural ligand-receptor interactions, such as the extracellular domain of the PD-1 protein (which can recognize PD-L1 / PD-L2 proteins), the extracellular domain of CD2 (recognizing CD58 / CD59), and the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86).

7. A chimeric antigen receptor (CAR) polypeptide as described in claim 5, wherein the transmembrane domain is selected from the transmembrane region of CD4, CD8, CD28, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor) or GITR; preferably, the transmembrane region of a type I single transmembrane molecule or a homologous multimer thereof; further preferably, it is the CD8 transmembrane domain.

8. A chimeric antigen receptor (CAR) polypeptide as described in claim 5, wherein the intracellular signaling domain is derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB) and FcεRIγ (FCERIG); preferably, derived from CD3ζ.

9. A nucleic acid sequence selected from: i) a nucleic acid sequence encoding the chimeric antigen receptor (CAR) polypeptide of any one of claims 4 to 8; or ii) a nucleic acid sequence complementary to i).

10. A nucleic acid construct comprising the nucleic acid sequence of claim 9; Preferably, the nucleic acid construct is a vector; More preferably, the nucleic acid construct is a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector, containing the above-mentioned nucleic acid sequence.

11. A lentiviral vector system, comprising the nucleic acid sequence of claim 9 and lentiviral vector auxiliary components.

12. A genetically modified cell, characterized in that The cell expresses the CAR molecule of the chimeric antigen receptor (CAR) polypeptide according to any one of claims 4-8, or contains the nucleic acid sequence according to claim 9, or contains the nucleic acid construct according to claim 10, or is infected with the lentiviral vector system according to claim 11; preferably, the cell is selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, red blood cells, and the T cells include αβT cells, γδT cells, and regulatory T cells.

13. A pharmaceutical composition or kit comprising a CAR molecule of a chimeric antigen receptor (CAR) polypeptide according to any one of claims 4 to 8, or a nucleic acid sequence according to claim 9, or a nucleic acid construct according to claim 10, or a lentiviral vector system according to claim 11, or a genetically modified cell according to claim 12; Preferably, the pharmaceutical composition or kit comprises a checkpoint inhibitor; More preferably, the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody or a combination thereof.

14. Use of the chimeric antigen receptor (CAR) polypeptide CAR molecule of any one of claims 4-8, or the nucleic acid sequence of claim 9, or the nucleic acid construct of claim 10, or the lentiviral vector system of claim 11, or the genetically modified cell of claim 12 in the preparation of any one or more of the following use products: (1) preparing T cells or NK cells; (2) enhancing the proliferation ability of T cells or NK cells; (3) improving the killing ability of T cells or NK cells.

15. Use of the chimeric antigen receptor (CAR) polypeptide CAR molecule of any one of claims 4 to 8, or the nucleic acid sequence of claim 9, or the nucleic acid construct of claim 10, or the lentiviral vector system of claim 11, or the genetically modified cell of claim 12 in the preparation of any one or more of the following products: (1) treating cancer; (2) inhibiting cytokine storm generated during cancer treatment; Preferably, the cancer is selected from adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer and thyroid cancer, and / or optionally wherein the cancer is a blood cancer or a solid tumor cancer.

16. The use according to claim 15, wherein the product further comprises a checkpoint inhibitor; Preferably, the checkpoint inhibitor comprises anti-PD-1 antibody, anti-PD-L1 antibody, PD-L1 recombinant protein and mutants thereof, PD-1 recombinant protein and mutants thereof, or a combination thereof.

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