Method for transforming chimeric antigen receptor based on phase separation and application thereof

Abstract

The invention discloses a method for transforming a chimeric antigen receptor based on phase separation and application thereof. The invention finds that the intracellular domain of CD28, the intracellular domain of Lck and the intracellular domain of PD-1 can be subjected to phase separation and regulate and control the activation of T cells, and researches on molecular mechanisms of phase separation of CD28 / Lck and CD28 / PD-1 find that two mutations of the intracellular domain of CD28 can inhibit phase separation of the intracellular domain of CD28 and PD-1 and do not influence phase separation of the intracellular domain of CD28 and Lck. The introduction of the two mutant CD28 intracellular domains in the design of the CAR-T is helpful for the CAR-T to resist the inhibition of PD-1, and the treatment effect can be further improved by combining the CAR-T with a PD-1 blocking antibody.

Description

Technical Field

[0001] 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, as well as recombinant genes designed based on this, vectors containing such genes, cells, and their applications. Background Art

[0002] T cells are an important part of the body's adaptive immunity. Through the TCR (T Cell Receptor) on their cell membrane, they can specifically recognize MHC (Major Histocompatibility Complex) molecules presenting specific antigen peptides on target cells, thereby initiating the activation pathway inside T cells and killing the target cells. In addition, the activation process of T cells is also jointly regulated by co-signal receptors and cytokines. Among them, co-signal 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 functions.

[0003] The regulatory role of co-signal receptor molecules in T cell activation has been widely applied in the clinical treatment of tumors and autoimmune diseases, which can be divided into immune blockade therapy and cell therapy. Among them, the combined therapy based on PD-1 and CTLA-4 antibodies has achieved good results in the clinical treatment of tumors. Chimeric Antigen Receptor (CAR) molecules designed based on these receptor molecules (CD28 and 4-1BB) have also been widely used in the engineering of T cells and tumor treatment. At the same time, it has been found that better clinical effects can be achieved by combining immune blockade therapy and cell therapy. However, antibody blockade sometimes does not work well, which may be due to ligand-independent signals of the receptor.

[0004] The disclosed patents (CN201711457252, CN201780059525, CN202111282362) involve combined therapies that combine PD-1 blockade and CAR-T. The CN201880045582 patent involves the application of two mutants of CD28 (mutation or deletion of YMNM and PRRP) in CAR-T or CAR-NK cancer treatment. Summary of the Invention

[0005] The present invention first provides a CD28 intracellular domain variant, and the variant contains the following mutation sites:

[0006] CD28-modi3: R195S and R201S;

[0007] CD28 - mRK: (185 - 188)SSSS;

[0008] CD28 - mBRS1: (162 - 167)SSSSS;

[0009] CD28 - mBRS2: (179 - 186)SSPGPTSS;

[0010] CD28 - mPRS1: (178 - 183)SRRSGS;

[0011] CD28 - mPRS2: (190 - 194)SYASS

[0012] CD28 - mdBRS1: Δ(162 - 167);

[0013] CD28 - mtPRS2: Δ(190 - 202);

[0014] or

[0015] CD28 - modi1: G182P, T184K, D196Q and A198T;

[0016] or any combination of the above sites, preferably, a combination of CD28 - modi3 or CD28 - mRK with the other above - mentioned sites.

[0017] 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 includes truncation, extension or conservative substitution.

[0018] In a specific embodiment of the present invention, the CD28 intracellular domain variant also optionally includes:

[0019] 1) Any truncation or extension, truncating or extending 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids at the C - or N - terminus based on the sequence of the CD28 intracellular domain (amino acids 162 - 202), and adding a tag or amino acids such as cysteine for labeling or separation at the C - or N - terminus also belongs to this type of variant. Those skilled in the art can understand that the above - mentioned tags or amino acids are removed when designing or preparing the CAR molecule;

[0020] 2) Any conservative mutation, which means that one amino acid is substituted by another amino acid within the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid;

[0021] Optionally, the variant has a sequence identity of greater than 95%, 96%, 97%, 98% or 99% with the CD28 intracellular domain (amino acids 162 - 202).

[0022] The present invention also provides a chimeric antigen receptor (CAR) that can specifically recognize tumor - associated antigens (TAAs) on cancer, and the CAR comprises the above - mentioned CD28 intracellular domain variant.

[0023] On the other hand, the present invention provides a chimeric antigen receptor (CAR) polypeptide, which comprises 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 enhances the resistance of CAR - T cells to PD - 1 inhibition.

[0024] In a specific embodiment of the present invention, the co - stimulatory signaling region comprises the CD28 intracellular domain variants mRK and modi3.

[0025] In a specific embodiment of the present invention, the TAA - binding region is selected from single - chain antibodies against tumor surface antigens, and the tumor surface antigens are 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 antibodies are selected from single - chain antibody fragments, single - chain Fv (scFv), single - chain Fab, single - chain Fab′, single - domain antibody fragments, single - domain multispecific antibodies, intracellular antibodies, nanobodies or single - chain immune factors;

[0026] 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 Taisheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96 and Glematumamab.

[0027] In another specific embodiment of the present invention, 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 (which recognizes CD58 / CD59), the extracellular domain of CD28 / CD152 (which recognizes CD80 / CD86), etc.

[0028] In another specific embodiment of the present invention, the transmembrane domain is selected from the transmembrane regions 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-pass transmembrane molecule or its homologous multimer; more preferably, the transmembrane domain of CD8.

[0029] The present invention also provides nucleic acid sequences selected from:

[0030] i) encoding the above-mentioned CAR molecule; or ii) a nucleic acid sequence complementary to i).

[0031] The present invention also provides a nucleic acid construct containing the above-mentioned nucleic acid sequence;

[0032] Preferably, the nucleic acid construct is a vector;

[0033] 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.

[0034] The present invention also provides a lentiviral vector system containing the above-mentioned nucleic acid sequence and lentiviral vector auxiliary components.

[0035] 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, red blood cells, and the T cells include αβ T cells, γδ T cells, and regulatory T cells.

[0036] The present invention also provides a pharmaceutical composition or a kit, which comprises the above-mentioned CAR molecule, the nucleic acid sequence containing the above, or the above-mentioned nucleic acid construct, or the above-mentioned lentiviral vector system or the above-mentioned genetically modified cell;

[0037] In a specific embodiment of the present invention, the pharmaceutical composition or the kit comprises a checkpoint inhibitor;

[0038] In a specific embodiment of the present invention, the checkpoint inhibitor includes an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody or a combination thereof.

[0039] 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 the preparation of the following product(s) for any one or more of the following uses: (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.

[0040] 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 the preparation of the following product(s) for any one or more of the following uses: (1) treating cancer; (2) inhibiting cytokine storm generated during cancer treatment;

[0041] 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 the cancer is a hematological cancer or a solid tumor cancer.

[0042] The present invention also provides a method for providing anti-tumor immunity in a subject suffering from 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.

[0043] 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.

[0044] In a specific embodiment of the present invention, it further comprises administering a checkpoint inhibitor to the subject.

[0045] In a specific embodiment of the present invention, the checkpoint inhibitor includes an anti-PD-1 antibody, an anti-PD-L1 antibody, a PD-L1 recombinant protein and its mutants, a PD-1 recombinant protein and its mutants, or a combination thereof.

[0046] Beneficial technical effects

[0047] In the present invention, it is found that the intracellular domain of CD28 can undergo phase separation with Lck and the intracellular domain of PD-1, and regulate the activation of T cells. By studying the molecular mechanism of the phase separation of CD28 / Lck and CD28 / PD-1, it is found that two mutations in the intracellular domain of CD28 can inhibit its phase separation with PD-1 and do not affect its phase separation with Lck. Introducing these two mutant intracellular domains of CD28 in the design of CAR-T helps CAR-T resist the inhibition of PD-1, and the therapeutic effect can be further improved by combining with PD-1 blocking reagents. Brief description of the drawings

[0048] Figure 1 : Phase separation of CD28 and Lck on a two-dimensional membrane ( Figure 1 A: Phase separation of the intracellular domain of the co-signaling receptor with the truncated Lck (upper) and the full-length Lck (lower) on a two-dimensional membrane; Figure 1 B: Analysis results of the phase separation of the intracellular domain of the co-signaling receptor with the truncated Lck; Figure 1 C: Analysis results of the phase separation of the intracellular domain of the co-signaling receptor with the full-length Lck).

[0049] Figure 2 : Phase separation of CD28, CD3ε and Lck in a solution system ( Figure 2 A: Co-localization of CD28 / pCD28 and CD3ε in the phase separation droplets; Figure 2 B: The phase separation droplets formed by CD28, CD3ε and Lck have good fluidity)

[0050] Figure 3 : PD-1 regulates the phase separation of CD28 and Lck ( Figure 3 A: pPD-1 inhibits the phase separation of Lck UD-SH3 and pCD28, Figure 3 B: pPD-1 inhibits the phase separation of Lck UD-SH3-SH2 and pCD28, PD-1 does not affect Lck UD-SH3 and Lck UD-SH3-SH2 and the phase separation of CD28)

[0051] Figure 4 : Double-phosphorylated PD-1 mediates the inhibition of pCD28 / Lck phase separation ( Figure 4A: The intracellular domain of doubly phosphorylated PD-1 inhibits the two-dimensional phase separation of pCD28 and Lck; Figure 4 B: Only phosphorylated PD-1 can be recruited by the pCD28 / Lck phase separation droplets; Figure 4 C: The intracellular domain of doubly phosphorylated PD-1 inhibits the three-dimensional phase separation of pCD28 and Lck

[0052] Figure 5 : The phase separation droplets of pCD28 / Lck recruit pPD-1 Figure 5 A: PD-1 is excluded from the pCD28 / Lck phase separation in the two-dimensional membrane system; Figure 5 B: pPD-1 is recruited by the pCD28 / Lck phase separation in the two-dimensional membrane system; Figure 5 C: PD-1 is excluded from the pCD28 / Lck phase separation in SLB-stimulated Jurkat cells; Figure 5 D: pPD-1 is recruited by the pCD28 / Lck phase separation in SLB-stimulated Jurkat cells

[0053] Figure 6 : The phase separation of PD-1 and CD28 is specific Figure 6 A: The two-dimensional phase separation of PD-1 with co-stimulatory molecules and CD3ε; Figure 6 B: The two-dimensional phase separation of pPD-1 with co-stimulatory molecules and CD3ε

[0054] Figure 7 : CD28 mutants weaken the phase separation of CD28 and PD-1 Figure 7 A: Confocal imaging of the phase separation of CD28 and its mutants with Lck and PD-1 respectively; Figure 7 B: Statistics of Lck aggregation mediated by CD28 and its mutants; Figure 7 C: Statistics of PD-1 aggregation mediated by CD28 and its mutants

[0055] Figure 8 : CD28 mutants resist the inhibition of pPD-1 on their phase separation with Lck Figure 8 A: PD-1 regulates the phase separation of phosphorylated CD28 and its mutants with Lck; Figure 8 B: Statistics of the size of the phase separation droplets

[0056] Figure 9 : CD28 mutants resist the ligand-independent inhibitory effect of PD-1 Figure 9 A: Three distribution patterns of PD-1 on the Jurkat-Raji interaction surface; Figure 9 B: Statistics of the proportions of the three distributions of PD-1 on the interaction surface between CD28 and its mutant cells and Raji cells;Figure 9 C: Co-culture of CD28 and its mutant cells with Raji cells expressing and not expressing PD-L1, and statistical analysis of residual activity based on the secretion of IL-2)

[0057] Figure 10 : CD28 mutants improve the ability of immune cells to resist PD-1 inhibition( Figure 10 A: CD28 mutation enhances the secretion of IL-2 by CAR-T cells under PD-L1 inhibition; Figure 10 B: Residual activity of CD28 and its mutant CAR-T cells under PD-L1 inhibition)

[0058] Figure 11 : CD28 mutants enhance the effect of combination of second-generation CAR-T and PD-L1 blockade

[0059] Figure 12: A: Intracellular domain of human CD28; B: CD28 variant of the present invention Detailed implementation manners

[0060] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0061] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation manners and not for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0062] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field of the present invention. Except for the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the technical field of the present invention and the description of the present invention, any methods, devices, and materials similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0063] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0064] Technical terms

[0065] The CAR molecule provided by the present invention is defined by the following formula:

[0066] SP–TAA–HG–TM–CSR–ISD;

[0067] or

[0068] SP–TAA–HG–TM–ISD–CSR;

[0069] wherein "SP" represents an optional signal peptide,

[0070] wherein "TAA" represents a TAA-binding region,

[0071] wherein "HG" represents an optional hinge domain,

[0072] wherein "TM" represents a transmembrane domain,

[0073] wherein "CSR" represents a co-stimulatory signaling region,

[0074] wherein "ISD" represents an intracellular signaling domain, and

[0075] wherein "–" represents a peptide bond or a linker.

[0076] Among them, the signal peptide is located at the extracellular end of the CAR molecule, and 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. Any signal peptide sequence in animal cells can be used on the CAR molecule. In the present invention, the signal peptide is optionally present, that is, the signal peptide can be used or not used.

[0077] Among them, the antigen recognition region recognizes tumor surface antigens. The antigen recognition region is preferably a single-chain antibody against tumor surface antigens. The tumor surface antigens are 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 single-chain antibody fragments, single-chain Fv (scFv), single-chain Fab, single-chain Fab′, single-domain antibody fragments, single-domain multispecific antibodies, intracellular antibodies, nanobodies, or single-chain immune factors.

[0078] 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.

[0079] Among them, the above antigen recognition region also includes 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), the extracellular domain of CD28 / CD152 (recognizing CD80 / CD86), etc.

[0080] Among them, the "hinge region" refers to the hydrophilic region between the antigen recognition domain and the transmembrane domain. In the present invention, the hinge region is optionally present, that is, it can be used or not used. The hinge region can use the hinge regions of various different antibodies or antigen receptors, especially the hinge regions of CD molecules. In a specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, IgG4, CD279 (PD-1).

[0081] Among them, the "transmembrane region" only needs to include a peptide capable of penetrating the cell membrane. The transmembrane region preferably used is the transmembrane region of CD molecules. In one embodiment, the transmembrane region can be selected, for example, from the transmembrane regions 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 its homologous multimer; in a preferred embodiment of the present invention, the CD8 transmembrane domain is adopted.

[0082] Among them, the "costimulatory signaling region" refers to a part of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for the effective response of lymphocytes to antigens. Examples of such molecules include 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 ligands that specifically bind to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Therefore, although the CAR of the present invention mainly uses a CD28 mutant as the costimulatory signal element, it can be used in combination with other costimulatory signal elements.

[0083] The preferred costimulatory signaling region of the present invention is a CD28 intracellular domain variant that enhances the resistance of CAR-T cells to PD-1 inhibition.

[0084] The CD28 intracellular domain in the present invention is positions 162-202 of the CD28 molecule (see Figure 12A and SEQ ID NO:1), and includes optionally possible expression or purification tags;

[0085] The mutation sites contained in the CD28 variant are:

[0086] CD28-modi3: R195S and R201S (SEQ ID NO:2);

[0087] CD28-mRK: (185-188)SSSS (SEQ ID NO:3);

[0088] CD28-mBRS1: (162-167)SSSSS (SEQ ID NO:4);

[0089] CD28-mBRS2: (179-186)SSPGPTSS (SEQ ID NO:5);

[0090] CD28-mPRS1: (178-183)SRRSGS (SEQ ID NO:6);

[0091] CD28-mPRS2: (190-194)SYASS (SEQ ID NO:7);

[0092] CD28-mdBRS1: Δ(162-167) (SEQ ID NO:8);

[0093] CD28-mtPRS2: Δ(190-202) (SEQ ID NO:9);

[0094] or

[0095] CD28-modi1: G182P, T184K, D196Q, and A198T (SEQ ID NO:10);

[0096] or any combination of the above sites.

[0097] For the specific mutation sites above, see ( Figure 12B ).

[0098] The above CD28 intracellular domain variants also optionally include:

[0099] 1) Any truncation or extension, truncating or extending 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids at the C- or N-terminus based on the sequence of the CD28 intracellular domain (amino acids 162-202), and adding a tag or amino acid such as cysteine for labeling or separation at the C- or N-terminus also belongs to this type of variant. Those skilled in the art can understand that the above tags or amino acids are removed when designing or preparing the CAR molecule;

[0100] 2) Any conservative mutation, which means that one amino acid is replaced by another amino acid within the same category. For example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid;

[0101] Optionally, the variant has a sequence identity greater than 95%, 96%, 97%, 98%, or 99% with the sequence of the CD28 intracellular domain (amino acids 162-202).

[0102] Among them, the "intracellular signaling domain" is derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (Fcgamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0103] In certain embodiments, the intracellular signaling domain is derived from CD3ζ (TCRζ, GenBank accession number BAG36664.1). The T cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as the T cell receptor T3 zeta chain or CD247 (cluster of differentiation 247), is a protein encoded by the CD247 gene in humans.

[0104] In the present invention, the term "identity" or "homology" generally refers to the proportion of nucleotide bases or amino acid residues in a candidate sequence that are the same as those in the corresponding sequence after sequence alignment and, if necessary, introducing gaps to achieve the maximum percentage of identity over the entire sequence and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions or insertions should 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 by sequence analysis software.

[0105] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid within the same category, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table:

[0106] Original residue Exemplary substitution Preferred conservative substitution Ala (A) Val (V); Leu (L); Ile (I) Val (V) Arg (R) Lys (K); Gln (Q); Asn (N); His (H) Lys (K) Asn (N) Gln (Q); His (H); Asp (D); Lys (K); Arg (R) Gln (Q) Asp (D) Glu (E); Asn (N) Glu (E) Cys (C) Ser (S); Ala (A) Ser (S) Gln (Q) Asn (N); Glu (E) Asn (N) Glu (E) Asp (D); Gln (Q) Asp (D) Gly (G) Ala (A) Ala (A) His (H) Asn (N); Gln (Q); Lys (K); Arg (R) Arg (R) Ile (I) Leu (L); Val (V); Met (M); Ala (A); Phe (F); Norleucine Nle Leu (L) Leu (L) Norleucine Nle; Ile (I); Val (V); Met (M); Ala (A); Phe (F) Ile (I) Lys (K) Arg (R); Gln (Q); Asn (N); His (H) Arg (R) Met (M) Leu (L); Phe (F); Ile (I) Leu (L) Phe (F) Trp (W); Leu (L); Val (V); Ile (I); Ala (A); Tyr (Y) Tyr (Y) Pro (P) Ala (A) Ala (A) Ser (S) Thr (T) Thr (T) Thr (T) Val (V); Ser (S) Ser (S) Trp (W) Tyr (Y); Phe (F) Tyr (Y) Tyr (Y) Trp (W); Phe (F); Thr (T); Ser (S) Phe (F) Val (V) Ile (I); Leu (L); Met (M); Phe (F); Ala (A); Norleucine Nle Leu (L) .

[0107] Nucleotide sequences and vectors

[0108] The polynucleotide sequences of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The present invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but have different nucleotide sequences.

[0109] The polynucleotide sequences of the present invention can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0110] The nucleic acid constructs provided by the present invention further include one or more regulatory sequences operably linked to the aforementioned polynucleotide sequences. The coding sequences of the CAR molecules of the present invention can be manipulated in various ways to ensure the expression of the protein. The nucleic acid constructs can be manipulated according to the differences or requirements of the expression vectors before inserting them into the vectors. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0111] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.

[0112] The regulatory sequence can 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 can be used in the present invention.

[0113] The regulatory sequence can also be a suitable leader sequence, the untranslated region of the mRNA that is important for translation in 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 can be used in the present invention.

[0114] Preferably, the nucleic acid construct is a vector.

[0115] Generally, the expression of the polynucleotide sequence encoding the CAR molecule is achieved by operably linking the polynucleotide sequence encoding the CAR to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0116] 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 plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Further, the vector is an expression vector. The expression vector can be provided to cells 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, herpesviruses, and lentiviruses. Generally, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0117] More preferably, the nucleic acid construct is a lentiviral vector containing an origin of replication, 3' LTR, 5' LTR, and the aforementioned polynucleotide sequence.

[0118] Constitutive promoter sequences can be used for the promoter, 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 leukosis 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, inducible promoters can also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked to the inducible promoter during the period of expression and turn off the expression when the expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0119] To evaluate the expression of the CAR molecule polypeptide or a portion thereof, the expression vector introduced into the cells can also contain either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population sought to be transfected or infected by the viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin, puromycin, and the like.

[0120] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0121] Methods for introducing genes into cells and for expressing genes in cells are known in the art. Vectors can be readily introduced into host cells by any method in the art, for example, mammalian, bacterial, yeast, or insect cells. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0122] 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 water-in-oil emulsions, micelles, mixed micelles, and liposomes.

[0123] 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. Selected genes can be inserted into vectors and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. 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, lentiviral vectors are used.

[0124] The present invention provides a lentiviral vector system, which contains 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 formed by virus packaging of the aforementioned nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line. The method for constructing the lentiviral vector system is a commonly used method in the art.

[0125] Cell therapy

[0126] The present invention also includes a class of cell therapies, in which T cells are genetically modified to express the CAR molecules described in the present invention (hereinafter referred to as CAR-T cells), and the CAR-T cells are injected into recipients in need thereof. The injected cells are capable of killing the tumor cells of the recipient.

[0127] The CAR-T cells of the present invention include TRUCK, universal CAR, self-driving CAR, Armored CAR, self-destructing CAR, conditional CAR, tagged CAR, TanCAR, dual CAR or sCAR.

[0128] TRUCK (T cells redirected for universal cytokine-mediated killing) co-expresses a chimeric antigen receptor (CAR) and an anti-tumor cytokine. Cytokine expression can be constitutive or induced by T cell activation. Targeting CAR specificity, the local production of pro-inflammatory cytokines recruits endogenous immune cells to the tumor site and may enhance the anti-tumor response.

[0129] Universal allogeneic CAR T cells are engineered to no longer express the endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thus preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0130] Self-driving CAR T cells co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing the tumor homing of the CAR T cells.

[0131] Engineered CAR T cells (Armored CAR) resistant to immunosuppression 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)), can be used in combination with immune checkpoint switch receptors, or can be used in combination with monoclonal antibodies that block immune checkpoint signaling.

[0132] Self-destructing CAR can be designed using RNA delivered by electroporation to encode the CAR. Alternatively, the inducible apoptosis of T cells can be achieved based on the binding of ganciclovir to thymidine kinase in genetically modified lymphocytes or the more recently described system of activating human caspase 9 by a small molecule dimer.

[0133] By default, the conditional CAR T cells are unresponsive or "off" until a small molecule is added to complete the pathway, enabling full transduction of Signal 1 and Signal 2, thereby activating the CAR T cells. Alternatively, the T cells can be engineered to express an adaptor-specific receptor that has affinity for a second antibody directed against the target antigen for subsequent anti-tumor immune responses mediated by the CAR-T cells, which can be either active or passive immune responses.

[0134] The labeled CAR T cells express the CAR plus the tumor epitope to which the existing monoclonal antibody binds. In intolerable adverse reactions, administration of the monoclonal antibody clears the CAR T cells and alleviates the symptoms without other off-tumor effects.

[0135] The tandem CAR (TanCAR) is composed of two single-chain variable fragments (scFv) in series fused to the intracellular domain of a T cell co-stimulatory molecule and the intracellular domain of CD3ζ. Activation of TanCAR T cells can be achieved when the target cell expresses one of the two targets.

[0136] The dual 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 the dual CAR T cells requires simultaneous recognition of two co-expressed targets on the tumor.

[0137] The safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR are only activated when encountering target cells that have the standard CAR target but lack the sCAR target.

[0138] In addition, the CAR-T-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific to the antigen-binding portion in the CAR molecule.

[0139] Treatable cancers can be non-solid tumors, such as hematological tumors, for example, leukemia and lymphoma.

[0140] The present invention provides genetically modified T cells or pharmaceutical compositions containing such genetically modified T cells, wherein the cells contain the aforementioned polynucleotide sequences, or contain the aforementioned nucleic acid constructs, or are infected with the aforementioned lentiviral vector system.

[0141] 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 relevant cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may comprise 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.

[0142] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease.

[0143] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein can be in the range of 10 4 to 10 9 cells / kg body weight, preferably in the range of 10 5 to 10 6 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered by infusion techniques well known in immunotherapy. The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the signs of the patient's disease and thus adjusting the treatment.

[0144] Administration of the subject composition can be effected in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally to a patient. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of T cells can be directly injected into a tumor, lymph node or site of infection.

[0145] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment can be combined with various radiotherapy agents, including: cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, and mycophenolic acid, etc. In a further embodiment, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

[0146] 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.

[0147] "Patient", "subject", "individual", etc. are used interchangeably herein and refer to a living organism that can elicit an immune response, such as a mammal. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species.

[0148] Optionally, the tumor is selected from one or more of leukemia or solid tumors.

[0149] Optionally, the tumor is selected from B cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0150] Materials and Methods

[0151] Antibody

[0152] The antibodies used in the present invention are purchased from various commercial suppliers:

[0153] 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, Abcam, ab245764), CD3ζ antibody (Santa Cruz Biotechnology, sc_1239), pZap70 antibody (Cell Signaling Technology, 2701), Zap70 antibody (Abcam, ab32410), pPLCγ1 antibody (Cell Signaling Technology, 14008), PLCγ1 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).

[0154] Chimeric molecule construction

[0155] All chimeric molecules were carried by lentiviral vectors. The entire chimeric molecule from the N-terminus to the C-terminus consisted of an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain. The four domains were connected in sequence by homologous recombination. The extracellular domain was the sequence of the CD19 single-chain antibody (clone number FMC63), and the signal peptide of CD8α was used. The intracellular domain was the intracellular domain of costimulatory molecules (CD137, CD28 and their mutants) connected in sequence with the intracellular domain of CD3ζ.

[0156] Jurkat cells

[0157] The relevant chimeric molecules were overexpressed in Jurkat cells by lentiviral infection. The successfully infected cells were sorted by flow cytometry and used for subsequent functional experiments. All Jurkat cells were cultured in RPMI1640 medium containing 100 U / mL streptomycin, 100 μg / mL penicillin, and 10% fetal bovine serum at 37 °C and 5% CO2.

[0158] Cell function experiment

[0159] 1×10 4 The corresponding 1×10⁶ Jurkat cells were seeded into a U-shaped 96-well culture plate 30 min in advance, and the corresponding number of Raji cells expressing or not expressing PD-L1 were added. The total volume was 100 μl, and the cells were cultured in an incubator at 37 °C for 16 - 24 h. In the PD-L1 blocking experiment, the Raji cells expressing PD-L1 were pre-incubated with the PD-L1 blocking antibody for 30 min, and then the corresponding number of second-generation CAR-Jurkat cells were added. The culture supernatant was collected and the secretion level of IL-2 was detected by ELISA. Each experiment was performed in triplicate parallel and three independent repeated experiments.

[0160] Cell imaging experiment

[0161] 1×10 4 The corresponding 1×10⁶ Jurkat cells were added into a 96-well optical imaging plate, and then the same amount of Raji cells expressing or not expressing PD-1 were added. After co-culturing for 30 min, imaging was performed using a confocal microscope. PD-1 and CD28 on the Jurkat-Raji interaction surface were analyzed by Image J.

[0162] For two-dimensional cell imaging, SUV containing biotin was used for membrane coating. Streptavidin was first linked on the two-dimensional membrane, and then biotin-labeled anti-CD3ε single-chain antibody, anti-CD28 antibody, and PD-L1 were linked. The corresponding Raji cells were added and incubated in an incubator for 30 min, then fixed with paraformaldehyde and subjected to corresponding immunofluorescence staining, and imaging analysis was performed by TIRF-SIM.

[0163] Peptides

[0164] Unless otherwise specified, all peptides used in the present invention were synthesized by GL Biochem (Shanghai, China).

[0165] Recombinant protein expression and labeling

[0166] Lck

[0167] 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 (3-509 a.a., Lck-wt; K273R&Y505F, Lck-open) were subcloned into the pHAGE vector. The pHAGE-Lck-wt or pHAGE-Lck-open plasmids were transiently transfected into Expi293F cells by polyethyleneimine (PEI) (Polysciences, 23966). After 48 hours of expression, the cells were harvested and lysed in 50 mM Na2HPO4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1X protease inhibitor mixture, 1 mM PMSF, 0.3% Triton X-100, pH = 8.0. The clarified lysate containing the recombinant protein was obtained by centrifugal filtration through a 0.22 μm membrane filter.

[0168] To express the Lck regulatory domain constructs, cDNAs encoding UD (3-60 a.a.), UD-SH3 (3-121 a.a.), UD-SH3-SH2 (3-226 a.a.), or SH3-SH2 (59-226 a.a.) and an N-terminal 10×His tag were subcloned into the pET28a vector and transduced into competent Escherichia coli BL21(DE3) strains. The recombinant BL21 was induced with 0.25 mM IPTG at 16 °C for 12 hours for the expression of the recombinant protein. The bacteria were harvested and lysed in 50 mM Na 2 HPO 4 4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1X protease inhibitor mixture, 1 mM PMSF, pH = 8.0. For the UD recombinant protein, the lysate was first treated by boiling at an additional 95 °C for 30 minutes. The clarified lysate containing the recombinant protein was obtained by centrifugal filtration through a 0.22 μm membrane filter.

[0169] To express the Lck kinase domain (245 - 501 a.a.), the encoding cDNA was inserted into the pFast Bac1-MBP vector, which has an N-terminal MBP, an HRV 3C site, and a 10×His sequence. Through the Bac to-Bac expression system, a recombinant baculovirus containing the expression plasmid was obtained. The baculovirus was produced in SF9 insect cells and used to infect BTI-Tn-5B1-4 insect cells for 48 hours. The cells were collected and lysed by an ultrasonic cell disruptor in 50 mM Na2HPO4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1X protease inhibitor mixture, 1 mM PMSF, pH = 8.0. The cleared lysate was incubated with amylose beads (Smart-lifesciences, SA026025). The MBP was removed with 3C protease at 4°C for 12 hours. The cleaved recombinant Lck kinase was obtained after centrifugation.

[0170] Intracellular domain of PD-1

[0171] The intracellular domain of PD-1 (193 - 288 a.a.) and the mutant cDNA sequence were inserted into the C-terminus of the pCold-GST plasmid and expressed as a fusion with GST and an 8×His-tag. The recombinant vector was transduced into competent Escherichia coli BL21(DE3). After culturing at 37°C, 0.5 M IPTG was added to induce at 15°C for 24 h. The cells were collected, lysed in PBS, and the supernatant of the lysate was collected. The target protein was enriched using a GST affinity column and digested overnight with 3C enzyme at 4°C. The flow-through was collected and purified using a Superdex75 molecular sieve. The target protein fraction was collected, concentrated, and then stored at -80°C.

[0172] PD-L1 protein

[0173] The extracellular domain cDNA of PD-L1 protein (19 - 238) was fused with a 10×His-tag and an AVI-tag and inserted into the pHAGE plasmid. The recombinant plasmid was transfected into Expi293F cells. After 48 h of expression, the supernatant of the culture medium was collected. After concentration, it 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, and then the excess biotin was removed by desalting and aliquoted for storage.

[0174] Fluorescent dye labeling

[0175] First, reduce the proteins and peptides used for fluorescence labeling with a 10-fold protein molar ratio of TCEP supplement, and then use a 5-fold protein molar ratio of maleimide (C5-maleimide Alexa-488, C5-maleimide Alexa-546, and add C2-maleimide Alexa-647, Thermo Scientific) and incubate at room temperature for 2 hours. Remove the excess dye by changing the buffer to PBS (Zeba spin desalting column, Thermo Scientific).

[0176] Phase separation on lipid-loaded bilayers

[0177] Small unilamellar vesicles (SUV)

[0178] Phospholipids (95% DOPC, 5% DGS-NTA-Ni 2+ and 0.1% DSPE-PEG5000 or 95% POPC, 5% DSPE-PEG3350-Biotin) are dried under a nitrogen stream in a 37 °C water bath. The dried lipid film is 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 is sonicated for 5 - 10 minutes and then repeatedly frozen and thawed until the solution becomes clear. The solution is centrifuged at 21,380 g for 60 minutes at 4 °C. Then collect the supernatant containing SUVs.

[0179] Supported lipid bilayers (SLB)

[0180] Wash the glass-bottom 96-well plate with 5% Hellmanex III and rinse thoroughly 3 times with MilliQ H2O. Then wash with 6M NaOH at 50 °C for 2 hours, then rinse thoroughly with MilliQ H2O, and then equilibrate with PBS for 30 minutes. Add 15 μl of freshly prepared SUVs and incubate at 37 °C for 1 hour to induce SLB formation. Then wash the SLBs 3 times with aggregation buffer (PBS containing 1% BSA) and incubate at room temperature for 30 minutes.

[0181] Membrane phase separation assay

[0182] Generally, incubate 2 μM His-tagged wild-type or truncated Lck with the preformed SLBs at room temperature for 1 hour. Remove the unbound proteins by washing three times with aggregation buffer. After incubating for another 30 minutes, add 2 μM His-tagged CD3ε or 10 μM CD3 molecules (CD3ε, CD3δ, CD3γ, and CD3ζ, without His-tag) to the glass wells and mix well to trigger cluster formation. Measure the occupied area and Feret diameter of the formed clusters using Image J.

[0183] Unless otherwise specified, all imaging experiments were performed on an Olympus FV1200 microscope equipped with a 60× oil immersion objective lens.

[0184] In-solution phase separation assay

[0185] The corresponding concentrations of CD3ε or PD-1 were pre-mixed with CD28, and then added to Lck or its various truncated proteins respectively and quickly mixed. After standing at room temperature for 1 h, imaging was performed using a confocal microscope, and the size of the clusters was analyzed by ImageJ. In the PD-1 recruitment experiment, CD28 and Lck were first formed into droplets, and then PD-1 or its mutants were added respectively and allowed to stand at room temperature for 30 min, and then imaging was performed using a confocal microscope, and the recruitment of PD-1 was analyzed by ImageJ.

[0186] Example

[0187] Example 1 CD28 can undergo phase separation with Lck on the two-dimensional membrane

[0188] As a second signal for T cell activation, co-signaling receptors can affect T cell function by regulating TCR activation. Our previous research results showed that during TCR activation, the CD3ε subunit in the TCR complex can undergo phase separation with the kinase Lck, thereby promoting TCR activation. However, the role of co-signaling receptors in this process remains unclear.

[0189] We used an in vitro reconstituted two-dimensional membrane system linked with the kinase Lck molecule to test several important co-signaling receptors, and found that only CD28 can undergo phase separation with Lck on the two-dimensional membrane ( Figure 1 ). At the same time, CD28 can be well recruited into the phase separation droplets of CD3ε / Lck; in the solution system, the phase separation droplets formed by CD28, CD3ε and Lck can also be well fused ( Figure 2 ).

[0190] Example 2 Phase separation droplets recruit pPD-1 (phosphorylated PD-1)

[0191] Studies have shown that CD28 is the main target of PD-1-mediated T cell inhibition. Therefore, we tested the effect of PD-1 on CD28 / Lck phase separation. The results showed that PD-1 did not significantly affect the phase separation of CD28 or pCD28 (phosphorylated CD28) from Lck. However, pPD-1 (phosphorylated PD-1) can mediate the dissolution of pCD28 / Lck phase separation droplets ( Figure 3)。The intracellular domain of PD-1 contains two phosphorylated sites: ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based switch motif). We separately tested the single-phosphorylated PD-1 at these two sites, and the results showed that single-phosphorylated PD-1 can help it be recruited by the pCD28 / Lck phase-separated droplets. However, only double-phosphorylated PD-1 can directly mediate the inhibition of pCD28 / Lck phase separation ( Figure 4 )。Meanwhile, we analyzed the distribution of PD-1 and pPD-1 on the reconstituted phospholipid membrane and inside cells respectively. The results showed that PD-1 was excluded from the pCD28 / Lck phase-separated droplets, while pPD-1 was recruited ( Figure 5 )。

[0192] Example 3 PD-1 can directly undergo phase separation with CD28

[0193] Further mechanism studies demonstrated that PD-1 can directly undergo phase separation with CD28, and pPD-1 mediates the dissolution of pCD28 / Lck phase-separated droplets by competitively binding to pCD28. Moreover, the phase separation between PD-1 and CD28 is specific and does not occur with other co-stimulatory receptor molecules ( Figure 6 )。

[0194] 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

[0195] Through molecular dynamics simulations, we explored the molecular mechanism of the phase separation between PD-1 and CD28. The results showed that PD-1 mainly binds to the C-terminal part of CD28, while Lck mainly binds to the N-terminal BRS region of CD28. Based on this, we designed two mutants (mRK and modi3) for the C-terminal region of CD28 and identified their phase separation with PD-1 and Lck on the two-dimensional membrane. The results showed that the two mutations do not affect the phase separation of CD28 and Lck, but greatly weaken the phase separation of CD28 and PD-1 ( Figure 7 )。

[0196] Example 5 Engineering chimeric antigen receptors based on phase separation

[0197] Immunotherapy based on PD-1 blockade has been widely used in tumor and other related diseases, but there are still some patients who do not respond. We speculate that the reason may be the non-ligand-dependent activity of PD-1. Some studies have shown that even in the absence of PD-L1, PD-1 will have a low level of phosphorylation; through cell co-culture, we also found that even when the target cells do not express PD-L1, some PD-1 will still be recruited to the cell-cell interaction surface. Compared with wild-type CD28, CD28-mRK and CD28-modi3 can inhibit this non-specific recruitment. At the same time, we also found that the two mutant CD28s can well resist the inhibition of pPD-1 on their separation from Lck( Figure 8 ). Functional experiments based on IL-2 secretion also showed that CD28-mRK and CD28-modi3 can better resist the non-ligand-dependent inhibitory effect of PD-1( Figure 9 ).

[0198] Next, we introduced these two mutations into the design of second-generation CAR-T (28z) and transfected them into Jurkat cells expressing PD-1 and not expressing PD-1 respectively, and then stimulated them by co-culturing with Raji-B cells expressing PD-L1. The results showed that the mutations did not affect the IL-2 secretion of T cells that do not express PD-1, but increased the IL-2 secretion of T cells that express PD-1. By analyzing the residual activity of T cells under PD-1 inhibition, we found that CAR-T containing these two CD28 mutants had higher residual activity( Figure 10 ). These results indicate that introducing mutations in CD28 in the design of CAR-T can greatly improve its ability to resist PD-1 inhibition.

[0199] We further detected the effect of CAR-T combined with PD-L1 blockade. Incubate the corresponding number of Raji cells expressing PD-L1 with PD-L1 blocking antibody in the incubator for 30 min, and then add the corresponding Jurkat cells expressing PD-1 transfected with second-generation CAR and culture them together. By detecting IL-2 in the culture supernatant, we found that CAR-T cells containing CD28-mRK and CD28-modi3 could secrete more IL-2, which was similar to the effect of CAR-T cells containing 4-1BB (CD137)( Figure 11 ). This indicates that by introducing mutations in CD28 in the design of second-generation CAR-T, the non-ligand-dependent inhibitory activity of PD-1 that appears in the combined treatment of CAR-T and PD-1 antibody can be compensated for.

Claims

1. CD28 intracellular domain variants, said mutants comprising 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 sites; preferably, comprising a combination of CD28-modi3 or CD28-mRK with the other above sites.

2. The CD28 intracellular domain variant according to claim 1, said variant further comprises: 1) Any truncation or extension, truncating or extending 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids at the C- or N-terminus based on the CD28 intracellular domain (amino acid positions 162-202) sequence; 2) Any conservative mutation, which means an amino acid is substituted by another amino acid within the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid; optionally, said variant has a sequence identity greater than 95%, 96%, 97%, 98% or 99% with the CD28 intracellular domain (amino acid positions 162-202) sequence.

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

4. Chimeric antigen receptor (CAR) polypeptide, said CAR comprising the CD28 intracellular domain variant according to any one of claims 1-3.

5. 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 said co-stimulatory signaling region comprises a region that enhances the resistance of CAR-T cells to PD-1 inhibition; preferably, said co-stimulatory signaling region comprises the CD28 intracellular domain variant according to any one of claims 1-3.

6. The chimeric antigen receptor (CAR) polypeptide according to claim 5, wherein the TAA-binding region is selected from single-chain antibodies against tumor surface antigens, and the tumor surface antigens are 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 antibodies are selected from single-chain antibody fragments, single-chain Fv (scFv), single-chain Fab, single-chain Fab', single-domain antibody fragments, single-domain multispecific antibodies, intracellular antibodies, nanobodies, or single-chain immune factors; 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 Taisheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, Retifanlimab, cBR96, and Glematumamab; The TAA-binding region in the CAR molecule also includes 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 (which recognizes CD58 / CD59), and the extracellular domains of CD28 / CD152 (which recognize CD80 / CD86).

7. The chimeric antigen receptor (CAR) polypeptide according to claim 5, wherein the transmembrane domain is selected from the transmembrane regions 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-pass transmembrane molecule or its homologous multimer; more preferably, the transmembrane domain of CD8.

8. The chimeric antigen receptor (CAR) polypeptide according to 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, it is derived from CD3ζ.

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

10. A nucleic acid construct, wherein the nucleic acid construct contains the nucleic acid sequence according to 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 nucleic acid sequence.

11. A lentiviral vector system, wherein the lentiviral vector system contains the nucleic acid sequence according to claim 9 and lentiviral vector auxiliary components.

12. A genetically modified cell, characterized in that, the cell expresses a 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 αβ T cells, γδ T cells, and regulatory T cells.

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

14. Use of a CAR molecule of the chimeric antigen receptor (CAR) polypeptide according to any one of claims 4-8, or the nucleic acid sequence according to claim 9, or the nucleic acid construct according to claim 10, or the lentiviral vector system according to claim 11, or the genetically modified cell according to claim 12, in the preparation of a product for any one or more of the following uses: (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. The CAR molecule of the chimeric antigen receptor (CAR) polypeptide according to any one of claims 4-8, or the nucleic acid sequence according to claim 9, or the nucleic acid construct according to claim 10, or the lentiviral vector system according to claim 11, or the genetically modified cell according to claim 12, for use in the preparation of a product for any one or more of the following uses: (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 hematological 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 an anti-PD-1 antibody, an anti-PD-L1 antibody, a PD-L1 recombinant protein and its mutants, a PD-1 recombinant protein and its mutants, or a combination thereof.

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