Co-expression of chimeric CD3 fusion protein and anti-CD3-based bispecific T cell activation elements

Abstract

Nucleic acids encoding chimeric CD3 fusion proteins and anti-CD3-based bispecific T cell activators (BiTA) elements are provided. Also provided are vectors, modified immune cells containing the nucleic acids, methods for their use, and methods for preventing tumors. BiTA secreted by CAB-T cells can simultaneously activate CAB-T cells in tumors and endogenous TCR complexes in unmodified T cells, exerting antitumor effects.

Description

Technical Field

[0001] Background Technical Field The present disclosure generally relates to immunotherapy, and in particular, the present disclosure relates to the co-expression of a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activation element.

Background Art

[0002] Related Art In recent years, immunotherapy has achieved unprecedented success in the complete remission rate of hematological malignancies. In 2017, two CAR-T products targeting CD19 successfully achieved commercialization and approval for the treatment of acute leukemia and non-Hodgkin lymphoma in children and young adults, respectively.

[0003] However, there are two major problems with immunotherapy for treating solid tumors. On the one hand, serious, even life-threatening clinical side effects associated with CAR-T therapy, mainly including cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, etc., are still a major risk in the clinical application of CAR-T therapy. Furthermore, CAR-T therapy has not yet demonstrated significant clinical efficacy in the clinical treatment of solid tumors.

[0004] WO2016070061 by Zhao et al. describes modified T cells that express a bispecific antibody and a chimeric ligand-modified activating receptor (CLEAR). However, the disclosure of Zhao is limited to the receptor / ligand targets PD1 / PD-L1 and CD27 / CD70, and the expression of PD1 or CD27 CLEAR.

[0005] WO2016 / 054520 by Kim et al. describes effector cells expressing a modified cell surface protein and the use of effector cells for the treatment of diseases. And in one of their embodiments, a combination therapy of effector cells with modified CD3e expression and a bispecific T cell engaging antibody (BiTE) is described. However, in Kim's method, CD3e and BiTE are not co-expressed in a single effector cell, and due to the short PK half-life and toxicity of BiTE, low-dose continuous infusion is required. The independent administration of these effector cells and BiTE does not produce a synergistic effect in the solid tumor microenvironment.

[0006] Therefore, there is still a need to develop a treatment regimen for inhibiting solid tumors that has good clinical efficacy and few clinical side effects. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] Summary An object of the present disclosure is to provide a treatment regimen for inhibiting solid tumors that has good clinical efficacy and few clinical side effects. MEANS FOR SOLVING THE PROBLEMS

[0008] A first aspect of the present disclosure provides a chimeric CD3 fusion protein (e.g., a chimeric CD3e fusion protein) and a DNA construct encoding an anti-CD3-based bispecific T cell activation element.

[0009] In a preferred embodiment, the chimeric CD3 is a fusion protein comprising one or more polypeptides that can be recognized by an anti-CD3 antibody and, optionally, one or more of a transmembrane domain (TM), a co-stimulatory domain, and a CD3 signal activation domain, such as a CD3ζ domain.

[0010] In a preferred embodiment, the anti-CD3-based bispecific T cell activation element is a fusion protein comprising one or more tumor antigen recognition domains and one or more anti-CD3 antibody fragments, and the anti-CD3 antibody fragments include, for example, single domain antibody sequences (VHH) targeting CD3, single chain antibody variable region sequences (scFv), and / or antigen-binding fragments (Fab). In a preferred embodiment, the chimeric CD3 fusion protein has a structure represented by the following formula I: L-EC-H-TM-C-CD3ζ (I) In this formula, L is absent or is a signal peptide sequence; EC is a polypeptide binding domain from or derived from the CD3e protein, which can be recognized by and binds to an anti-CD3 antibody, and is a polypeptide binding domain; The polypeptide binding domain is also referred to as the recognition binding domain of the anti-CD3 antibody; The polypeptide binding domain is also referred to as a subunit of the recognition binding domain of the anti-CD3 antibody; H is absent or is a linker or hinge region; TM is a transmembrane domain; C is absent or is a co-stimulatory signaling molecule; CD3ζ is absent or is a cytoplasmic signaling sequence derived from CD3ζ; Each "-" is independently a linker peptide or a peptide bond.

[0011] In another preferred embodiment, L is the signal peptide of a protein selected from the following group: CD8, GM-CSFR (DNA sequence number SEQ 1, AA sequence number SEQ 2), CD4, CD137, or a combination thereof.

[0012] In another preferred embodiment, the polypeptide binding domain is the CD3e extracellular region or a part thereof that can be recognized by an anti-CD3 antibody.

[0013] In another preferred embodiment, the anti-CD3 antibody is selected from the following group: scFV (single-chain antibody), single-domain antibody sequence (also known as nanobody or VHH), diabody, or a variant thereof, or a combination thereof.

[0014] In another preferred embodiment, the clone of the anti-CD3 antibody includes L2K (DNA sequence number SEQ 19, AA sequence number SEQ 20), UCHT1, OKT3, F6A, I2C, or a combination thereof. In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to an anti-CD3 antibody that may be present as a segment of a bispecific antibody.

[0015] In another preferred embodiment, the EC comprises or consists of positions 1 to 104 of wild-type or mutant CD3e protein, and its amino acid sequence is shown in SEQ ID NO: 4.

[0016] In another preferred embodiment, H is a linker or hinge region of a protein selected from the following group: CD8 (DNA sequence number SEQ 5, AA sequence number SEQ 6), CD28, (DNA sequence number SEQ 57, AA sequence number SEQ 58), CD137, or a combination thereof.

[0017] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: CD28 (DNA sequence number SEQ 59, AA sequence number SEQ 60), CD3 epsilon, CD45, CD4, CD5, CD8 (DNA sequence number SEQ 7, AA sequence number SEQ 8), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0018] In another preferred embodiment, C is a costimulatory signaling molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0019] In another preferred embodiment, C comprises a costimulatory signaling molecule derived from 4-1BB (DNA SEQ ID NO: 9, AA SEQ ID NO: 10), and / or a costimulatory signaling molecule derived from CD28 (DNA SEQ ID NO: 61, AA SEQ ID NO: 62).

[0020] In another preferred embodiment, CD3ζ is a cytoplasmic signaling sequence represented by AA SEQ ID NO: 12.

[0021] In another preferred embodiment, the DNA construct is expressed in cis or in a fusion form with a safety switch protein, and the proteins that can function as a safety switch include inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, Claudin 18.2, or a combination thereof.

[0022] In another preferred embodiment, the anti-CD3-based bispecific T cell activation element (BiTA) has a structure represented by the following formula II: L’-T1-B1-B2-T2 (II) In this formula, L’ is absent or is a signal peptide sequence; T1 is absent or is a tag element; B1 is a tumor antigen recognition region or a CD3 antigen recognition region; B2 is a CD3 antigen-binding antibody fragment or a tumor antigen recognition region; T2 is absent or is a tag element; Each "-" is independently a linker peptide or a peptide bond.

[0023] In another preferred embodiment, L’ is a signal peptide of a protein selected from the group consisting of: CD8, GM-CSFR, CD4, CD137, or combinations thereof.

[0024] In another preferred embodiment, the tag element comprises a tag protein, a fluorescently labeled protein or an enzymatically labeled protein.

[0025] In another preferred embodiment, the tag proteins include the FLAG protein (DNA sequence number SEQ 13, AA sequence number SEQ 14), and the His protein (DNA sequence number SEQ 35, AA sequence number SEQ 36).

[0026] In another preferred embodiment, B1 is a tumor antigen recognition region and B2 is a CD3 antigen recognition region.

[0027] In another preferred embodiment, the tumor antigen recognition region comprises one or more receptor or ligand binding domains; an antibody fragment comprising a single domain antibody sequence (VHH) and / or a single chain antibody variable region sequence (scFv); and / or a TCR sequence.

[0028] In another preferred embodiment, the tumor antigen is selected from the following group: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosine kinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostain, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3 or a combination thereof.

[0029] In another preferred embodiment, the tumor antigen recognition region targets CAIX and / or HER2.

[0030] In another preferred embodiment, the tumor antigen recognition region is a VHH antibody (DNA sequence number SEQ 17, AA sequence number SEQ 18) that targets CAIX.

[0031] In another preferred embodiment, the tumor antigen recognition region is a single-chain antibody (DNA sequence number SEQ 65, AA sequence number SEQ 66) that targets HER2.

[0032] In another preferred embodiment, the CD3 antigen-binding antibody fragment is a single-domain antibody sequence (VHH), a single-chain antibody variable region sequence (scFv), or an antigen-binding fragment (Fab) that targets CD3.

[0033] In another preferred embodiment, the BiTA is a secreted BiTA. In another preferred embodiment, the BiTA targets either CAIX (DNA sequence number SEQ 21, AA sequence number SEQ 22) or HER2 (DNA sequence number SEQ 49, AA sequence number SEQ 50).

[0034] In another preferred embodiment, the secreted BiTA can be autocrine and / or paracrine.

[0035] In another preferred embodiment, the cell type secreting the secreted BiTA can be a T cell, NK cell, macrophage, B cell, red blood cell, or a combination thereof.

[0036] In another preferred embodiment, the cell type secreting the secreted BiTA is a T cell. In another preferred embodiment, the BiTA can bind to chimeric CD3e.

[0037] In another preferred embodiment, BiTA can bind to the T cell receptor (TCR) complex.

[0038] In another preferred embodiment, the TCR is derived from T cells according to the fifth aspect and / or non-modified T cells.

[0039] In another preferred embodiment, a nucleic acid molecule encoding a chimeric CD3 fusion protein and a nucleic acid molecule encoding a bispecific T cell activation element are provided separately. Advantageously, the nucleic acid molecule encoding the chimeric CD3 fusion protein and the nucleic acid molecule encoding the bispecific T cell activation element are co-expressed in the same immune cell.

[0040] A second aspect of the present invention provides a vector characterized by comprising a nucleic acid molecule. In another preferred embodiment, the vector is selected from the group consisting of lentivirus, adenovirus, and retroviral vectors.

[0041] A third aspect of the present invention provides a genetically modified immune cell (e.g., T cell) characterized by expressing the above nucleic acid molecule. In an embodiment as required, the immune cell is modified to express a chimeric CD3 fusion protein and a bispecific T cell activation element, and the nucleic acid molecule encoding the chimeric CD3 fusion protein and the nucleic acid molecule encoding the bispecific T cell activation element are not provided on the same DNA construct.

[0042] In another preferred embodiment, the T cell is derived from a human or non-human mammal. In another preferred embodiment, the T cell further comprises other chimeric antigens.

[0043] A fourth aspect of the present disclosure provides a composition characterized by comprising a fusion protein and BiTA.

[0044] In another preferred embodiment, the fusion protein in the composition is located in the extracellular region of the T cell membrane.

[0045] In another preferred embodiment, the BiTA in the composition is an autocrine, paracrine or exogenous BiTA.

[0046] In another preferred embodiment, the composition is expressed in the form of a fusion protein of Formulas I and II with a 2A protein, the structural formula thereof being I-2A-II or II-2A-I, and the sequence of 2A includes one of T2A (DNA sequence number SEQ 23, AA sequence number SEQ 24), P2A, F2A or E2A or a combination thereof.

[0047] In another preferred embodiment, the I-2A-II structure is a sequence targeting CAIX (DNA sequence number SEQ 25, AA sequence number SEQ 26) or a sequence targeting HER2 (DNA sequence number SEQ 49, AA sequence number SEQ 50).

[0048] In another preferred embodiment, the II-2A-I structure is a sequence targeting CAIX or HER2 (DNA sequence number SEQ 67, AA sequence number SEQ 68).

[0049] In another preferred embodiment, the I-2A-II or II-2A-I structure is expressed in cis or in a fusion form with a safety switch protein, and the proteins capable of functioning as a safety switch include inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, Claudin 18.2, or a combination thereof.

[0050] In another preferred embodiment, the composition is expressed in the form of a combination of an IRES sequence and a fusion protein of Formulas I and II, the structural formula thereof being I-IRES-II or II-IRES-I, and the IRES is an internal ribosome entry site within the nucleotide sequence.

[0051] In another preferred embodiment, the IRES functions to initiate amino acid translation of the downstream gene.

[0052] In another preferred embodiment, the I-IRES-II or II-IRES-I structure is expressed in cis or in a fusion form with a safety switch protein, and the proteins that can function as safety switches include inducible caspase 9 (iCasp9), CD19, CD20, EGFR, HER2, CD30, CD19, c-Met, Claudin 18.2, or combinations thereof.

[0053] The fifth aspect of the present disclosure provides a non-naturally occurring T cell population. The above T cells are present in the T cell population at a ratio C1 of 10% or higher based on the total number of T cells in the T cell population.

[0054] In another preferred embodiment, C1 is 10% or higher, preferably C1≧20%, more preferably C1≧30%.

[0055] In another preferred embodiment, BiTA and / or BiTA-secreting T cells C2 are also present in the T cell population.

[0056] The sixth aspect of the present disclosure provides a composition comprising (a) the above genetically modified T cells and / or the above T cell population, and (b) a pharmaceutically acceptable carrier, diluent and / or excipient.

[0057] The seventh aspect of the present disclosure relates to the use of the above genetically modified T cells, T cell population, and / or composition in the preparation of a medicament for the prevention and / or treatment of cancer or tumor or for use in the following methods.

[0058] The eighth aspect of the present disclosure provides a method for preventing or treating a disease, the method comprising administering an appropriate amount of the above genetically modified T cells, T cell population, and / or composition to a subject in need of treatment.

[0059] In another preferred embodiment, the disease is cancer or a tumor. In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, and combinations thereof.

[0060] In another preferred embodiment, the hematological tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and combinations thereof.

[0061] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, urinary tract tumor, thyroid cancer, and combinations thereof.

[0062] In another preferred embodiment, the solid tumor is selected from the following group: ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, liver cancer, endometrial cancer, or combinations thereof. In another preferred embodiment, the method further comprises administering an appropriate amount of a cytokine or drug compound secreted by the stimulating cells and compositions thereof to enhance the responsiveness of immune cells.

[0063] In one embodiment, the method further comprises administering dasatinib to the subject. In another preferred embodiment, the immune cells include the above-mentioned T cells, T cell populations and / or compositions, as well as endogenous T cells, NK cells, macrophages and B cells.

[0064] A ninth aspect of the present disclosure is a method for reducing the toxicity of immune cells modified with a chimeric CD3e fusion protein and a bispecific T cell activation element, the method comprising administering dasatinib; and the use of dasatinib in the preparation of a medicament for reducing the toxicity of immune cells modified with a chimeric CD3e fusion protein and a bispecific T cell activation element.

[0065] Within the scope of the present disclosure, it will be understood that various technical features of the present disclosure described above and various technical features specifically described below (for example, in the examples) can be combined with each other to constitute new or preferred technical solutions.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0067] Detailed Description The present disclosure relates to an immunotherapy regimen for inhibiting tumors, particularly solid tumors, including the use of T cells that express a chimeric CD3 fusion protein, preferably a chimeric CD3e fusion protein, together with an anti-CD3 antibody-based bispecific T cell activator (BiTA) similar to a bispecific T cell engager antibody (BiTE). The chimeric CD3 fusion protein and the BiTA having a CD3 antigen recognition site bind to each other and perform the functions of activating T cells and targeting tumor cells that express tumor-associated antigens (TAAs). The present disclosure also provides CAB constructs and CAB-modified T cells (CAB-T) that express a chimeric CD3 and an anti-CD3 antibody-based bispecific T cell activator. The BiTA secreted by the CAB-T cells simultaneously activates the CAB-T cells and the endogenous TCR complexes of unmodified and modified T cells in tumor tissue, exerts the anti-tumor effect of CAB-T, and mobilizes the anti-tumor effect of unmodified T cells, thereby ensuring the effectiveness of this CAB-T technology for clinical applications. The chimeric CD3 constructs expressed by CAB-T and BiTA operate synergistically to exert their anti-tumor effects: the activation of the chimeric CD3 element is dependent on the BiTA secreted by CAB-T, and the secretion of BiTA further stimulates CAB-T to secrete BiTA by activating the chimeric CD3 element. Thus, immune cell activation and anti-tumor effects are localized in the tumor microenvironment, ensuring the safety advantages of the clinical application of this CAB-T technology.

[0068] Term Explanation Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0069] As used herein, the term "about", when used with respect to a specifically recited value, means that the value can vary by up to 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values between them (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0070] As used herein, the term "comprising" or "include" can be non - limiting, semi - limiting, or limiting. In other words, this term also includes "consisting essentially of" or "consisting of".

[0071] The term "administering" refers to the physical introduction of the products of the present disclosure to a subject, for example, by injection or infusion, using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration.

[0072] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins or antigen - binding fragments thereof that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain includes a heavy - chain variable region (abbreviated herein as VH) and a heavy - chain constant region. The heavy - chain constant region includes three constant domains designated CH1, CH2, and CH3. Each light chain includes a light - chain variable region (abbreviated herein as VL) and a light - chain constant region. The light - chain constant region includes the constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity - determining regions (CDRs), which are interspersed with more highly conserved regions called framework regions (FRs). Each of VH and VL contains three CDRs and four FRs, which are arranged in the following order from the amino - terminus to the carboxyl - terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

[0073] It should be understood that the amino acid names in this specification are given by the international one-letter English symbols, and the three-letter English abbreviations corresponding to these amino acid names are Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y) and Val(V), respectively.

[0074] Chimeric antigen receptor (CAR) The structure of the chimeric antigen receptor (CAR) is a fusion protein based on the intracellular segment domain of the TCR complex CD3ζ and the intracellular activator domain from the co-stimulatory signal CD28 or 4-1BB. Advantageously, T cells modified to express CAR can bind to target antigens in an MHC-independent manner, and thus, the activation of T cells does not depend on the presentation of antigens by MHC. This type of CAR is known as a second-generation CAR structure, and the two CAR-T drugs approved in 2017 belong to this structural type.

[0075] T cell receptor (TCR) The T cell receptor (TCR) is the most complex receptor in the human body, and its extensive signaling within T cells is determined by the interaction of six different receptor subunits with each other. Two chains, TCRα and TCRβ, together recognize a complex composed of a polypeptide-major histocompatibility complex, and the subunits that transmit TCR signals are collectively called CD3 and include one heterodimer formed by CD3ε and CD3γ, one heterodimer formed by CD3ε and CD3δ, and one CD3ζ homodimer. All subunits of the TCR, except for CD3ζ, are type I transmembrane proteins and have immunoglobulin domains. The four different CD3 subunits in the TCR receptor complex have a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs) and can receive a total of 20 tyrosine phosphate groups when the TCR receptor complex is activated. Transgenic mouse experiments have shown that changes in the proline-rich region of the intracellular segment of CD3ε or changes in the higher-order structure of CD3ε play extremely important regulatory roles in the delivery of intact TCRs. It has been demonstrated that TCR activity can be modulated by binding a ligand to TCRαβ to stabilize the arrangement of CD3 subunits, ligand-independent TCR oligomerization, and binding to cholesterol.

[0076] TRuC structure TCR 2The novel T cell therapy platform, TRuC™, developed in-house, is a chimeric antigen receptor consisting of an antibody-based target antigen recognition sequence and a TCR receptor subunit. The TRuC structure can reprogram a complete TCR complex that recognizes tumor antigens. Unlike the CAR structure, the TRuC structure can be incorporated into the TCR complex to exert its function. TRuC-T has the same anti-tumor activity as second-generation CAR-T. In addition, since TRuC-T cells do not have an additional co-stimulatory signaling domain (CD28 or 4-1BB), they release cytokines at a significantly lower level than CAR-T cells. TRuC-T shows anti-tumor activity in both hematological tumor transplantation models and solid tumor transplantation models. In addition, TRuC-T shows strong anti-tumor activity compared to CAR-T in multiple tumor models.

[0077] T cell antigen conjugate (TAC) The TAC (T cell antigen conjugate) technology platform by Triumvira can induce a stronger anti-tumor response with lower toxicity than CAR-T by modulating the endogenous TCR of T cells. The TAC structure consists of three parts: 1. an extracellular antigen-binding region, 2. a TCR mobilization region of a CD3 single-chain antibody, and 3. a CD4 / CD8 co-receptor binding region. Preclinical experiments have demonstrated that the TAC-T technology can specifically bind to tumor cells and cause cytotoxicity, and the activation of TAC-T is similar to the activation of normal T cells, thus preventing the production of a large number of cytokines. In a mouse tumor transplantation model, TAC-T shows better activity than CAR-T against either solid tumors or hematological tumors. In addition, TAC-T has a high ability to infiltrate the tumor microenvironment of solid tumors.

[0078] Bispecific T cell engager antibody (BiTE) Blinatumomab, a bispecific T cell engager (BiTE) drug targeting CD19 and developed by Amgen, USA, was approved by the FDA in 2014 for the clinical treatment of acute leukemia. This antibody consists of two parts: an scFv that recognizes the CD19 antigen and an scFv that recognizes the TCR complex (CD3e). When it recognizes the target antigen CD19 within tumor cells, the BiTE antibody activates T cells and induces tumor cell death by inducing oligomerization of the endogenous TCR of T cells using the anti-CD3 scFv portion. The way BiTE treats tumors is similar to that of TRuC and TAC technologies, all of which cause activation of the endogenous TCR in T cells. Theoretically, these three have an equivalent ability to activate the endogenous TCR signal and all may be very useful for mobilizing and redirecting T cells to treat solid tumors. However, due to the poor safety caused by systemic administration of BiTE drugs, the extremely short half-life of BiTE drugs in vivo, and similar issues, the use of BiTE in achieving desirable efficacy in the clinical treatment of solid tumors has not yet been demonstrated.

[0079] Bispecific T cell activator (BiTA) structure As used herein, the terms "bispecific T cell activator structure", "bispecific T cell activation element", "BiTA", "bispecific T cell activator", and "-BiTA" refer to an anti-CD3-based bispecific T cell activator structure that comprises the following two parts: (i) one or more tumor antigen recognition regions, which are antibody fragments comprising a single domain antibody sequence (VHH), single-chain antibody variable region sequence (scFv), antigen-binding fragment (Fab) and / or T cell receptor (TCR) sequence, such as a receptor or ligand-binding domain, etc., that recognize a tumor antigen, and (ii) one or more CD3 antigen recognition regions, which are antibody fragments such as a single domain antibody sequence (VHH), single-chain antibody variable region sequence, or antigen-binding fragment (Fab) that target CD3.

[0080] In another preferred embodiment, the anti-CD3-based bispecific T cell activation element has a structure represented by the following formula II: L’-T1-B1-B2-T2 (II) In this formula, L’ is a signal peptide sequence that is absent or derived from a protein selected from the following group: CD8, GM-CSFR, CD4, CD137, or a combination thereof.

[0081] T1 is absent or is a tag element, which optionally includes a tag protein, a fluorescently labeled protein or an enzymatically labeled protein. Preferably, the tag proteins include the FLAG protein (DNA sequence number SEQ 13, AA sequence number SEQ 14), and the His protein (DNA sequence number SEQ 35, AA sequence number SEQ 36).

[0082] B1 is a tumor antigen recognition region, and this tumor antigen recognition region may contain, as needed, a receptor or ligand binding domain, a single domain antibody sequence (VHH), and / or a single-chain antibody variable region sequence (scFv), and / or an antibody Fab, and / or a T cell receptor (TCR) sequence, and these / these can recognize tumor antigens selected from the following group: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostate specific antigen, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosine kinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostain, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2-ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3 or a combination thereof. Preferably, the tumor antigen recognition region B1 targets the tumor antigens CAIX and / or HER2.

[0083] In another preferred embodiment, the tumor antigen recognition region B1 is a VHH antibody (DNA sequence number SEQ 17, AA sequence number SEQ 18) that targets CAIX.

[0084] In another preferred embodiment, the tumor antigen recognition region B1 is a single-chain antibody (DNA sequence number SEQ 65, AA sequence number SEQ 66) derived from trastuzumab that targets HER2.

[0085] B2 is a CD3 antigen recognition region and, optionally, is a single-domain antibody sequence (VHH) and / or antibody Fab and / or single-chain antibody variable region sequence (scFv) that targets CD3. In a preferred embodiment, the CD3 antigen-binding antibody fragment can be derived from CD3 Ab clones such as L2K, UCHT, OKT3, F6A, SP34.

[0086] Optionally, the positions of B1 and B2 may be reversed. T2 is either absent or is a tag element which, optionally, contains a tag protein, a fluorescently labeled protein or an enzymatically labeled protein. Preferably, the tag proteins include a FLAG protein (DNA sequence number SEQ 13, AA sequence number SEQ 14), and a His protein (DNA sequence number SEQ 35, AA sequence number SEQ 36).

[0087] Each "-" is independently a linker peptide or a peptide bond. CD3e protein Both the "CD3e protein" and "CD3e" described herein refer to the human CD3e protein.

[0088] The "extracellular region of the CD3e protein" described herein refers to amino acids 1 to 104 of the CD3e protein sequence exemplified by SEQ ID NO:4.

[0089] The protein sequence includes an amino acid sequence having a homology of 60% or more, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with an amino acid sequence.

[0090] Chimeric CD3e fusion protein The "chimeric CD3e fusion protein", "CD3e fusion protein" and "chimeric CD3e protein" described herein refer to a fusion protein expressed in T cells having a structure represented by the following formula I: L-EC-H-TM-C-CD3ζ (I) In this formula, each "-" is independently a linker peptide or a peptide bond; L is an optional signal peptide sequence; EC is a polypeptide binding domain from or derived from the CD3e protein, which can be recognized by an anti-CD3 antibody and binds to the anti-CD3 antibody; The polypeptide binding domain is also referred to as the recognition binding domain of the anti-CD3 antibody; The polypeptide binding domain is also referred to as a subunit of the recognition binding domain of the anti-CD3 antibody; H is a linker or hinge region as required; TM is a transmembrane domain; C is either absent or a costimulatory signaling molecule; CD3ζ is either absent or a cytoplasmic signaling sequence derived from CD3ζ.

[0091] In another preferred embodiment, the polypeptide binding domain is the CD3e extracellular region exemplified by SEQ ID NO:4, or a portion thereof that can be recognized by an anti-CD3 antibody.

[0092] In another preferred embodiment, the anti-CD3 antibody is selected from the following group: scFV (single-chain antibody), single-domain antibody sequence (also known as nanobody), diabody, antibody Fab or its variant, or a combination thereof.

[0093] In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to an anti-CD3 antibody that can exist as a segment of a bispecific antibody.

[0094] In another preferred embodiment, the hinge region is the CD8 hinge, and its amino acid sequence is SEQ ID NO:3.

[0095] In another preferred embodiment, the transmembrane region is CD8 TM, and its amino acid sequence is SEQ ID NO:8.

[0096] Chimeric CD3 and anti-CD3-based bispecific T cell activators, modified T cells, CAB-T As described herein, the “chimeric CD3 and anti-CD3-based bispecific T cell activator-modified T cells”, “CAB-T cells”, “CAB-T technology”, “CAB structure”, “CAB-T”, “-CAB-T” and “-CAB” refer to T cells modified to co-express both a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activator. Preferably, the modified T cells contain a construct having the following structure: (i) chimeric CD3e in a CAB structure containing at least the following four components: CD3e extracellular region, CD8 hinge region and transmembrane region, 4-1BB intracellular region, and CD3 intracellular region; (ii) an anti-CD3-based bispecific T cell activator (BiTA) containing at least the following two parts: a receptor or ligand binding domain, which is an antibody fragment containing a single domain antibody sequence (VHH), antibody Fab fragment, single-chain antibody variable region sequence (scFv) or T cell receptor (TCR) sequence that recognizes and binds to a tumor antigen, and a variable region sequence that recognizes the CD3 antigen in the TCR complex.

[0097] As described above, the amino acid sequence of the CD3e extracellular region is shown in SEQ ID NO: 4, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 10, and the amino acid sequence of CD3ζ is shown in SEQ ID NO: 12.

[0098] Other preferred synthetic gene sequences are shown in Table 1 below:

[0099]

Table 1-1

[0100]

Table 1-2

[0101] CAIX CAIX is a transmembrane protein expressed in various solid tumor cells. The main function of CAIX is to maintain intracellular pH homeostasis under hypoxic conditions commonly found in solid tumors. The expression of CAIX in tumor cells is considered a marker protein for the hypoxic state of the tumor environment and poor patient prognosis. Common types of tumors that express CAIX include cervical cancer, kidney cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, bladder cancer, and the like. In normal tissues, CAIX is mainly expressed in epithelial cells of the bile duct and small intestine, as well as gastric epithelial cells, etc. However, unlike tumor cells, CAIX expressed in normal tissues is mainly localized in the cytoplasm. Therefore, CAIX is an ideal therapeutic target for targeted therapies including cell therapy.

[0102] HER2 HER2 is one of the most studied targets for tumor immunotherapy and is generally expressed in tissues such as breast cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, urothelial cancer, ovarian cancer, and lung cancer. Trastuzumab, a monoclonal antibody drug targeting HER2, has greatly improved the quality of life and extended the lifespan of patients with HER2-positive breast cancer. However, there are still many patients with HER2-positive tumors who do not respond to trastuzumab or develop resistance to trastuzumab. Therefore, there is still a large market demand for the development of new therapies targeting HER2. Currently, there are reports on CAR-T drugs targeting HER2. Among them, Steven A Rosenberg reported severe toxicity and side effects in a clinical trial of a third-generation CAR-T drug targeting HER2. Such drugs cause dyspnea and severe immune cell infiltration into the lungs, thus leading to patient death. Therefore, at the development stage of drugs targeting HER2 cells, such drugs must be designed to emphasize drug safety.

[0103] Composition The present disclosure provides a composition or formulation comprising T cells modified to co-express both a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activator (i.e., CAB-T cells), together with a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the composition is a liquid formulation. Preferably, the composition is an injectable. Preferably, the concentration of CAB-T cells in the composition is 1×10 3 ~1×10 8 cells / ml, more preferably 1×10 4 ~1×10 7 cells / ml.

[0104] In one embodiment, the composition may include a buffer, such as, for example, neutral buffered saline, sulfate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, and mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0105] Therapeutic applications The present disclosure encompasses the therapeutic applications of T cells modified to co-express both a chimeric CD3 fusion protein and an anti-CD3-based bispecific T cell activator (i.e., CAB-T cells). Cells transduced with a vector containing the nucleic acid construct of the present disclosure can target tumor cell markers, while autocrine or paracrine BiTA (secreted by the modified T cells) can synergistically activate T cells and significantly improve their lethal efficiency against tumor cells by eliciting a T cell immune response.

[0106] Accordingly, the present disclosure also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, the method comprising the step of administering CAB-T cells.

[0107] The cancers that can be treated include not only tumors that are not angiogenesis or substantially not angiogenesis, but also angiogenesis tumors. Cancers can include non-solid tumors (e.g., blood tumors such as leukemia and lymphoma) or solid tumors. The types of cancers that can be treated with the nucleic acid constructs and modified T cells of the present disclosure include carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas and melanomas, etc., but are not limited thereto. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0108] Blood cancers are cancers of the blood or bone marrow. Examples of blood (hematogenous) cancers include leukemias, polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin lymphoma (indolent and high-grade types), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myeloproliferative disorders, such as acute leukemias (e.g., acute lymphocytic leukemia, acute myeloblastic leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, granulocyte-monocyte leukemia, monocytic leukemia and erythroleukemia, etc.), chronic leukemias (e.g., chronic myeloblastic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia, etc.).

[0109] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid regions. Solid tumors can be either benign or malignant. Different types of solid tumors are named according to the cell type that forms them (e.g., sarcomas, carcinomas, and lymphomas, etc.). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0110] The CAB-modified T cells of the present disclosure can also be used as a vaccine for ex vivo immunotherapy and / or in vivo treatment in mammals. Preferably, the mammal is a human.

[0111] Regarding ex vivo immunotherapy, at least one of the following is performed in vitro prior to administration of the cells to a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAB into the cells, and / or iii) cryopreserving the cells.

[0112] Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAB disclosed herein. The CAB-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the CAB-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic to the recipient.

[0113] In addition to the use of cell-based vaccines for ex vivo immunotherapy, the present disclosure also provides compositions and methods for in vivo immunotherapy for eliciting an immune response against an antigen in a patient.

[0114] The present disclosure provides a method for treating a tumor, the method comprising administering to a subject a therapeutically effective amount of the CAB-modified T cells of the present disclosure.

[0115] The CAB-modified T cells of the present disclosure can be administered in the form of a pharmaceutical composition, either alone or in combination with a diluent and / or other components, such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present disclosure can comprise a population of target cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers, such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, and mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0116] The pharmaceutical compositions of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration will depend on factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0117] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the exact amount of the composition of the present disclosure to be administered can be determined by a physician taking into account the age, weight, tumor size and degree of infection or metastasis of the patient (subject), as well as individual differences in condition. The pharmaceutical composition containing the T cells described herein is administered at a dose of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It is generally shown that administration can be carried out at a dose of cells / kg body weight (including all integer values within these ranges). The T cell composition can also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). A person of ordinary skill in the medical field can readily determine the optimal dosage and treatment regimen for an individual patient by monitoring the signs of the patient's disease and thus adjusting the treatment.

[0118] The composition can be administered to a subject by any conventional method, including by inhalation, injection, oral administration, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intrathecally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell composition of the disclosure is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the disclosure is preferably administered by i.v. injection. The T cell composition can be injected directly into a tumor, lymph node or site of infection.

[0119] In certain embodiments of the present disclosure, using the methods described herein, or other methods known in the art for expanding T cells to therapeutic levels, the activated and expanded cells are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, treatment with cytarabine (also known as ARA-C) or natalizumab for patients with MS, or treatment with efalizumab for patients with psoriasis, or other treatments for patients with PML. In further embodiments, the T cells of the present disclosure can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject can receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present disclosure after transplantation. In one further embodiment, the expanded cells are administered before or after surgery.

[0120] The dosage of the above-described treatments administered to a patient will vary depending on the exact nature of the condition being treated and the recipient undergoing the treatment. Dosage ratios for administration to humans can be carried out according to accepted conventions in the art. Typically, 1×10 6 ~1×10 10 modified T cells of the present disclosure can be administered to a patient, e.g., by intravenous reinfusion.

[0121] The technical solutions of the present disclosure have the following beneficial effects: 1. The present disclosure provides an immunotherapy regimen for inhibiting tumors, particularly solid tumors, namely, the combined use of T cells expressing BiTA and a chimeric CD3 fusion protein. The chimeric CD3 fusion protein and BiTA bind to each other and perform the functions of activating T cells and targeting tumor cells.

[0122] 2. The present disclosure also provides a CAB technology such that, through the chimeric expression of CD3 and BiTA in T cells, CAB-T cells target tumor tissue. BiTA secreted by CAB-T cells simultaneously achieves the activation of CAB-T cells and the activation of the endogenous TCR complex of unmodified T cells in tumor tissue, exerts the anti-tumor effect of CAB-T itself, and mobilizes the anti-tumor effect of unmodified T cells, thereby ensuring the effectiveness of CAB-T clinical applications.

[0123] 3. Since the activation of chimeric CD3 depends on BiTA secreted by CAB-T cells, a small amount of BiTA released by CAB-T cells in tumor tissue can stimulate CAB-T to release more BiTA in the local tumor microenvironment, which advantageously guarantees the safety of CAB-T clinical applications.

[0124] 4. CAB-T can achieve better activation in solid tumor tissue, achieve the maximum anti-tumor effect at the tumor site, achieve the same safety and effectiveness as local administration of the tumor, and has great advantages and potential compared with second-generation CAR-T in the clinical treatment of solid tumors.

[0125] The present disclosure is further illustrated by specific examples below. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example

[0126] Example 1 Design of CAB and Its Control Structures 1.1 Structural Design of CD3e-BBζ, the First CAIX-BiTA, the First CAIX-CAB, and CAIX-TRuC in the Control Group To verify the antitumor activity of CAB-T, the inventors first designed four structures in the first group: CD3e-BBζ (DNA sequence number SEQ 15, AA sequence number SEQ 16), CAIX-BiTA (DNA sequence number SEQ 21, AA sequence number SEQ 22), CAIX-CAB (DNA sequence number SEQ 25, AA sequence number SEQ 26), and CAIX-TRuC (DNA sequence number SEQ 31, AA sequence number SEQ 32) using a nanobody (VHH) targeting CAIX in the experimental group. To distinguish from the CAIX-CAB structure of the second group described later, the inventors named the CAIX-BiTA and CAIX-CAB of this group the first CAIX-BiTA and the first CAIX-CAB, respectively. Among these, the first CAIX-BiTA is a tagless BiTA, the first CAIX-CAB contains a CD3e-BBζ and CAB structure without a labeled BiTA, and CAIX-TRuC is a control structure using the platform technology of TCR 2 Therapeutics. The specific structures of the above constructs are shown in Figure 1.

[0127] 1.2 Structural design of tERBB2, CD3e-BBζ, CAIX-BiTA, CAIX-CAB, CAIX-ζ, CAIX-BBζ, and CAIX-28ζ in the experimental group of nanobodies (VHH) targeting CAIX In another experimental group, the inventors used VHH targeting CAIX to create a second group of structures, including truncated ERBB2 (tERBB2, used as a negative control, containing four extracellular domains of ERBB2, a transmembrane region, and a FLAG tag) (DNA sequence number SEQ 33, amino acid sequence number SEQ 34), CD3e-BBζ (DNA sequence number SEQ 15, amino acid sequence number SEQ 16), CAIX-BiTA (DNA sequence number SEQ 37, amino acid sequence number SEQ 38), CAIX-CAB (DNA sequence number SEQ 39, amino acid sequence number SEQ 40), CAIX-ζ (first-generation CAR structure targeting CAIX) (DNA sequence number SEQ 41, amino acid sequence number SEQ 42), CAIX-BBζ (second-generation CAR structure containing the 4-1BB co-stimulatory domain) (DNA sequence number SEQ 45, amino acid sequence number SEQ 46), and CAIX-28ζ (second-generation CAR structure containing the CD28 co-stimulatory domain) (DNA sequence number SEQ 47, amino acid sequence number SEQ 48), for a total of 7 structures. In the structures of this group, all structures carry a FLAG tag, and all secreted BiTA antibodies have a His tag. Inclusion of the His tag facilitates subsequent detection of BiTA secretion levels. Details of these structures are shown in Figure 2.

[0128] 1.3 Structural design of tERBB2, CD3e-BBζ, HER2-BiTA, HER2-CAB, HER2-ζ, HER2-BBζ, and HER2-28ζ in the experimental group of single-chain antibodies targeting HER2 In the third experimental group, the inventors tested the antitumor activity of the CAB platform using a single-chain antibody (scFv) targeting HER2. The variable region sequence of this single-chain antibody is derived from the antibody drug Herceptin (trastuzumab). In this example, the inventors designed the structures of the third group, including a total of seven constructs: truncated ERBB2, CD3e-BBζ, HER2-CAB (DNA sequence number SEQ 49, AA sequence number SEQ 50), HER2-ζ (DNA sequence number SEQ 51, AA sequence number SEQ 52), HER2-BBζ (DNA sequence number SEQ 53, AA sequence number SEQ 54), and HER2-28ζ, and a second-generation CAR construct (DNA sequence number SEQ 55, AA sequence number SEQ 56). In the structures of this group, all constructs except HER2-ζ carry a FLAG tag, and all secreted BiTA antibodies also carry a His tag. Details are shown in Figure 3.

[0129] Example 2 Lentiviral packaging of CAB and its control constructs In the present disclosure, the inventors prepared CAB-T cells using a lentivirus as a vector. First, the inventors prepared a lentiviral vector carrying the gene encoding CAB and its control constructs. The specific procedures for packaging the lentivirus are as follows.

[0130] 1) Seed 1×10 7 HEK 293T cells into a 10-cm culture plate, add 10 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C), mix the cells well, and incubate overnight at 37°C.

[0131] 2) The next day, when the cell confluence of HEK 293T (ATCC, CRL-3216) reached approximately 90%, replace the medium with serum-free DMEM.

[0132] 3) Prepare a plasmid complex. In this preparation, the amounts of various plasmids are 8 μg of plasmid DNA, 4 μg of psPAX2, and 2 μg of pMD2g, respectively. Dissolve these in 1 mL of opti-MEM (Gibco, 31985-070), add 42 μL of PEI (Polysciences, 24765-2), and shake while vortexing for 20 seconds. After leaving it at room temperature for 15 minutes, gently add the mixture along the side to the HEK293T medium and keep the culture at 37°C.

[0133] 4) After culturing the cells for 4 hours, remove the medium, wash once with PBS (Hyclone, SH30256.01), and add fresh pre-warmed DMEM medium with 2% FBS again.

[0134] 5) After 48 hours and 72 hours of transfection, collect the supernatant respectively, centrifuge at 2000 g for 5 minutes, and discard the precipitate. Filter the supernatant through a 0.25 μm filter (Sartorius, 16541-K), then add PEG 8000 (Sigma, 89510-1KG-F) at a final concentration of 5% and NaCl (Sigma, S5150-1L) at a final concentration of 0.15 M, mix vigorously, and leave at 4°C overnight.

[0135] 6) Centrifuge the virus supernatant at 2000 g at 4°C for 20 minutes, remove the supernatant, dissolve the virus pellet in 50 - 100 μL of PBS, and freeze at -80°C.

[0136] Example 3 Preparation of Modified T Cells with CAB and Its Control Structure After completion of the preparation of the lentiviral vector carrying the CAB structure, the lentiviral vector can be used to infect immune cells to complete the preparation of CAB-T cells. The specific procedure for preparing CAB-T cells is as follows.

[0137] 1) Commercial PBMC (Saily Bio, SLB-HP050B) cells were cultured at an initial cell density of 1×10 6 / mL using X-VIVO 15 (LONZA, 04-418Q) containing 5% human blood albumin (GRIFOLS, 20% human blood albumin).

[0138] 2) Anti-CD3 / CD28 beads (Miltenyi biotec, 130-091-441) were added at a cell:bead ratio of 3:1, and 1000 IU / mL of IL-2 (Si Huan Sheng Wu, SFDA approval number: S10970016) was added to activate T cell expansion.

[0139] 3) After 48 hours of cell activation, the T cells were infected by adding an appropriate amount of virus and 12 μg / mL of protamine (Sigma, P4005).

[0140] 4) After 24 hours of lentivirus infection, the cell suspension was aspirated, and fresh X-VIVO 15 medium was added at a concentration of 1×10 6 cells / mL.

[0141] 5) The cell density was observed daily, and a T cell culture solution containing 1000 IU / mL of IL-2 was added when appropriate to maintain the T cell density at approximately 1×10 6 cells / mL, and expansion was continued for 5 - 10 days to complete the preparation of CAR-T cells.

[0142] Example 4 Positive frequency assay of CAB-T cells After the preparation of CAB-T and its control group cells was completed, the infection efficiency was determined for subsequent activity analysis. Specifically, the method for detecting the CAB-T positive frequency using the FLAG antibody is as follows.

[0143] 1) 3 - 5×10 5Collect cells, add 200 μL of FACS buffer (PBS containing 1% FBS) to each flow cytometry tube, and centrifuge the mixture at 300 g for 5 minutes; add biotin-CAIX (sino biological, 10107-H02H) to the CAIX-Truc sample at a final concentration of 100 nM, and incubate the mixture at 4°C for 20 minutes; add biotin-HER-2 (ACRO, HE2-H82E2) to the Her2-Truc sample at a final concentration of 100 nM, and incubate the mixture at 4°C for 20 minutes; discard the supernatant, add 200 μL of Fixation / Permeabilization solution (BD bioscience, 554715), and incubate the mixture at 4°C for 20 minutes.

[0144] 2) Centrifuge the mixture at 300 g for 5 minutes; remove the supernatant, add 200 μL of 1× Perm / Wash buffer (BD bioscience, 554715), resuspend the mixture, centrifuge at 400 g for 5 minutes, and wash twice.

[0145] 3) Remove the centrifuged supernatant, add 100 μL of anti-Flag antibody diluted 1:1000 with FACS buffer to each sample, mix the cells well, and incubate at 4°C for 30 minutes.

[0146] 4) After incubation, add 1 mL of FACS buffer to each flow cytometry tube, and centrifuge the mixture at 400 g for 5 minutes.

[0147] 5) Remove the centrifuged supernatant, add 1 mL of FACS buffer, resuspend the mixture, and centrifuge at 400 g for 5 minutes.

[0148] 6) Remove the centrifuged supernatant, add 100 μL of SA-PE (Invitrogen, S866) diluted 1:250 with FACS buffer to each sample, mix the cells well, and incubate at 4 °C for 30 minutes in the dark; after incubation, add 1 mL of FACS buffer to each flow cytometry tube, centrifuge the mixture at 300 g for 5 minutes; remove the centrifuged supernatant and repeat the washing twice.

[0149] 7) Subject the samples to a flow cytometer for detection. Results: In the first experimental group, since the FLAG tag is carried in the CD3e-BBζ structure and the first CAIX-CAB structure, the FLAG antibody can be used to detect the positive frequency of T cells modified by the corresponding structure. On the other hand, the biotin-labeled CAIX protein can be used to detect the positive frequency of T cell transduction for T cells modified by CAIX-TruC. However, since there is no suitable tag in the first CAIX-BiTA structure, the positive frequency of T cells modified by this structure cannot be detected. However, from the subsequent results, it can be determined that the positive frequency of T cells modified by the first CAIX-BiTA can meet the requirements of experimental analysis. The detection results are shown in Figure 4.

[0150] In the second and third experimental groups, the inventors designed a FLAG tag for each structure, and the positive frequency of the corresponding modified T cells can be determined by labeling and detection with the FLAG antibody (for the first-generation structure HER2-ζ without the FLAG tag, biotinylated HER2 was used to detect the transduction efficiency). NT represents non-transduced T cells, and NT was used as the negative control group. The test results are shown in Figures 5 and 6. The difference in infection efficiency between different samples is within the tolerance limit.

[0151] From the above, it can be seen that the positive frequency of transduction for each group of modified T cells designed by using the structures disclosed in this disclosure and the structures disclosed in the prior art meets the requirements of experimental analysis.

[0152] Example 5 Antigen-dependent cytokine release assay of CAB-T When CAB-T cells are co-cultured with tumor cells, CAB-T can recognize and activate the target antigen on the surface of tumor cells, and release a large number of inflammatory cytokines by activation. Based on this, the level of cytokines released by activated CAB-T cells was detected by enzyme-linked immunosorbent assay (ELISA) in this example.

[0153] The detection procedure of ELISA is as follows. 1) Effector cells at 1×10 5 and target cells at 1×10 5 were seeded at 200 μL / well. A 96-well cell culture plate was co-cultured overnight, centrifuged at 300 g for 5 minutes, and 150 μL of supernatant / well was transferred to a new 96-well cell culture plate using a multi-channel pipette, and cytokines were detected using IFN-γ (Invitrogen, 88-7316-88) / IL-2 (Invitrogen, 88-7025-88) / TNF-α (Invitrogen, 88-7346-88) assay kits respectively.

[0154] 2) A human anti-IFN-γ / IL-2 / TNF-α antibody was coated on the ELISA plate one day before. The human anti-IFN-γ / IL-2 / TNF-α antibody was diluted with PBS (1:250), 100 μL of the antibody was added to each well, and the ELISA plate was sealed at 4°C overnight using a microplate sealer.

[0155] 3) Plate washing: The liquid in the plate was quickly removed, and 200 μL / well of washing buffer was added using a multi-channel pipette, and this plate washing was repeated 5 times.

[0156] 4) 200 μL of 1×ELISA / ELISASPOT diluent was added to each well, covered using a microplate sealer, and shielded at room temperature for 60 minutes.

[0157] 5) Plate washing: The liquid in the plate was rapidly removed, and 200 μL / well of washing buffer was added with a multi-channel pipette. This plate washing was repeated 5 times.

[0158] 6) Human IFN-γ ELISA standards were prepared, and eight gradients (in pg / mL): 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 were set.

[0159] 7) Standards and samples were added to the ELISA plate at 100 μL / well. Both the samples and standards were also diluted to the desired concentration with 1×ELISA / ELISASPOT diluent, covered with a microplate sealer, and incubated at room temperature for 2 hours.

[0160] 8) Plate washing: The liquid in the plate was rapidly removed, and 200 μL / well of washing buffer was added with a multi-channel pipette. This plate washing was repeated 4 times.

[0161] 9) Human IFN-γ / IL-2 / TNF-α detection antibody was diluted with PBS (1:250), 100 μL of the antibody was added to each well, the ELISA plate was sealed with a microplate sealer, and incubated at room temperature for 1 hour.

[0162] 10) Streptavidin HRP conjugate was prepared by dilution with PBS (1:250), added to the ELISA plate at 100 μL / well, the plate was covered with a microplate sealer, and incubated at room temperature for 30 minutes.

[0163] 11) Plate washing: The liquid in the plate was rapidly removed, and 200 μL / well of washing buffer was added with a multi-channel pipette. This plate washing was repeated 5 times.

[0164] 12) The TMB substrate was warmed to room temperature 30 minutes before, and added to the ELISA plate at 100 μL / well. After reacting at room temperature for 5 - 10 minutes, 50 μL / well of stop solution was added.

[0165] 13) The absorbance was read with a microplate reader at a detection wavelength of OD = 450 nm.

[0166] 14) A standard curve was calculated according to the concentration of the standard substance and the OD value, and the concentration of the sample to be tested was calculated according to the standard curve. The plot was created using GraphPad Prism mapping software.

[0167] Results: In the first experimental group, CAIX-CAB-T cells or their control group cells were co-cultured with CAIX + HEK 293T cells or CAIX - HEK293T cells respectively, and the release levels of inflammatory cytokines IL-2 and IFN-γ in the supernatant were detected. The results are shown in Figure 7. When CAIX-CAB-T and its control group were co-cultured with CAIX - HEK 293T cells respectively, none of the immune cells showed significant cytokine release. When CAIX-CAB-T and its control group were co-cultured with CAIX + HEK 293T cells, all T cells modified by the first CAIX-BiTA, the first CAIX-CAB, and CAIX-TRuc showed co-culture time-dependent cytokine release levels. The cytokines accumulated after 48 hours of co-culture were significantly higher than the cytokine levels accumulated after 24 hours of co-culture. When unmodified T cells and T cells modified by CD3e-BBζ were co-cultured with CAIX + HEK 293T cells respectively, no significant release of IL-2 and IFN-γ cytokines was detected. In addition, unexpectedly, it was found that the level of cytokines released by the first CAIX-CAB-T cells was significantly higher than that of the first CAIX-BiTA-T cells after 48 hours of co-culture. T cells modified by CD3e-BBζ and T cells modified by the first CAIX-BiTA were mixed at a ratio of 1:1, and then CAIX +After co - culturing with HEK 293T cells, it was found that the cytokine release levels were equivalent to those of the first CAIX - CAB - T cells. The above results indicated that CAIX - CAB - T cell activation depends on the CAIX antigen, and that BiTA and CD3e - BBζ synergistically promote T cell activation. It was also demonstrated that CAB - T cells have an equivalent in vitro activation ability compared to the control group of TRuC - T cells.

[0168] In the second experimental group, CAIX - CAB - T and their control group cells were co - cultured with CAIX + MB - 231 or CAIX - MB - 231 cells respectively (the CAIX expression levels are shown in Figure 8.A), and then the release levels of the inflammatory cytokines IL - 2, IFN - γ and TNF - α in the supernatant were detected. The results are shown in Figure 8. When CAIX - CAB - T cells and their controls were co - cultured with CAIX - MB - 231 cells respectively, none of the cells showed significant cytokine release. After co - culturing CAIX - CAB - T and its control group with CAIX + MB - 231 cells for 48 hours, the T cells modified by CAIX - BiTA, CAIX - CAB, CAIX - BBζ, CAIX - 28ζ and CAIX - ζ had various levels of activation. From the data, it can be seen that CAIX - CAB - T and CAIX - BiTA, and the T cells structurally modified by the first and second generation CARs, have almost the same release ability in IFN - γ and TNF - α. On the other hand, regarding IL - 2 release, CAIX - CAB - T was weaker than the second generation CAR cells but slightly stronger than CAIX - BiTA and the first generation CAR - modified T cells. The results of the second experimental group indicated the dependence of CAIX - CAB - T cell activation on the CAIX antigen and the difference in cytokine release compared to the second generation CAR, that is, the IFN - γ and TNF - α released by CAIX - BiTA - T and CAIX - CAB - T are substantially equivalent to those released by the second generation CAR, and CAIX - BiTA - T and CAIX - CAB - T are weaker than the second generation CAR regarding IL - 2 release.

[0169] Example 6 Upregulation of Antigen-Dependent T Cell Activation Markers in CAB-T When CAB-T cells are co-cultured with tumor cells, CAB-T can recognize and activate the target antigen on the surface of tumor cells. The expression levels of T cell activation marker proteins on the cell membrane surface, including CD137, CD25, CD27 and the like, are significantly upregulated. The cell proliferation ability indicated by the expression level of Ki67 is increased, and the killing ability of T cells indicated by CD107a is also enhanced. Based on this, in this example, the above staining method and flow cytometry were used to detect the changes in the expression levels of the above-mentioned membrane surface proteins in activated CAB-T cells.

[0170] The specific cell staining procedure was as follows: 1) 1×10 5 effector cells and 1×10 5 target cells were seeded at 200 μL / well. A 96-well cell culture plate was co-cultured overnight, centrifuged at 300 g for 5 minutes, 200 μL of FACS buffer was added to each well, and centrifuged at 300 g for 5 minutes.

[0171] 2) The centrifuged supernatant was removed, the cells were resuspended by adding 200 μL of FACS buffer, and centrifuged at 300 g for 5 minutes.

[0172] 3) The antibodies were diluted with FACS buffer (100 μL / well): BV421 Mouse Anti-Human CD3 (BD Bioscience, 562426) diluted 1:500 PE Mouse Anti-Human CD137 (BD Bioscience, 555956) diluted 1:200 APC Mouse Anti-Human CD27 (BD Bioscience, 561786) diluted 1:200 PE-cy7 Mouse Anti-Human CD25 (BD Bioscience, 557741) diluted 1:200.

[0173] 4) Remove the centrifuged supernatant, add 100 μL of antibody to each well, mix, and incubate in the dark at 4 °C for 30 minutes.

[0174] 5) Add 200 μL of FACS buffer to each well, centrifuge at 300 g for 5 minutes, and remove the supernatant.

[0175] 6) Remove the centrifuged supernatant and repeat step 2.5. 7) Discard the supernatant, add 200 μL of Fixation / Permeabilization solution (BD bioscience, 554715), and incubate at 4 °C for 20 minutes.

[0176] 8) Centrifuge at 300 g for 5 minutes. Remove the centrifuged supernatant, add 200 μL of 1×Perm / Wash buffer (BD bioscience, 554715) for resuspension, and centrifuge at 400 g for 5 minutes.

[0177] 9) Wash twice and dilute the antibody FITC mouse anti-Flag (Biolegend, 637318) 1:1000 with FACS buffer.

[0178] 10) Centrifuge at 400 g for 5 minutes. Remove the centrifuged supernatant and wash twice.

[0179] 11) Perform flow cytometry detection using FSC / SSC gating to obtain the desired lymphocyte population (PBMC), select the CD3 BV421 + and Flag FITC + cell populations to obtain living CAR-T cells, and then gate by using PBMC not transduced with virus as a standard substance to obtain the percentage of CAR-T cell CD137 PE + cells.

[0180] Results: In the first experimental group, CAIX-CAB-T cells or their control group cells were co-cultured with CAIX + HEK 293T cells or CAIX - HEK293T respectively, and the changes in the expression levels of CD137 and CD107a on the surface of the T cell membrane were detected. The results are shown in Figure 9. When CAIX-CAB-T and its control group were co-cultured with CAIX - HEK 293T cells, the expression levels of CD137 and CD107a in CAB-T and its control group immune cells did not change significantly. After CAIX-CAB-T and its control group were co-cultured with CAIX + HEK 293T cells for 24 hours respectively, the expression levels of CD137 and CD107a in T cells modified by the first CAIX-BiTA, the first CAIX-CAB and CAIX-TRuC were significantly up-regulated, and the first CAIX-CAB-T showed a much higher up-regulation level compared with the first CAIX-BiTA. When unmodified T cells and T cells modified by CD3e-BBζ were co-cultured with CAIX + HEK 293T cells respectively, no significant up-regulation of CD137 and CD107a was detected. T cells modified by CD3e-BBζ and T cells modified by the first CAIX-BiTA were mixed at a ratio of 1:1, and then co-cultured with CAIX + HEK 293T cells for 24 hours. After that, the expression levels of CD137 and CD107a were found to be significantly higher than those of CD3e-BBζ-T and the first CAIX-BiTA-T co-cultured with CAIX + HEK 293T individually. The above results showed that CAIX-CAB-T cell activation depends on the CAIX antigen, and that BiTA and CD3e-BBζ synergistically promote T cell activation. It was also demonstrated that CAB-T cells have an in vitro activation ability equivalent to that of the control group TRuC-T cells.

[0181] In the second experimental group, CAIX-CAB-T cells and their control group cells were co-cultured with CAIX + MB-231 cells or CAIX- After co - culturing with MB - 231 for 48 hours each, the changes in the expression levels of CD137, CD25, CD27, and Ki67 on the surface of the T - cell membrane were detected. The results are shown in Figure 10. CAIX - CAB - T and its control group were CAIX - When co - cultured with MB - 231 cells respectively, the expression levels of CD137, CD25, CD27, and Ki67 in CAIX - CAB - T and its control group immune cells did not change significantly. CAIX - CAB - T and its control group were CAIX + After co - culturing with MB - 231 cells, the expression levels of CD137, CD25, CD27, and Ki67 in T cells modified by CAIX - BiTA, CAIX - CAB, the first - generation CAR, and the second - generation CAR were significantly up - regulated. T cells modified by tERBB2 and CD3e - BBζ were CAIX + When co - cultured with MB - 231 cells respectively, a significant up - regulation of CD137, CD25, CD27, and Ki67 was not detected. The above results indicate that CAIX - CAB - T cell activation depends on the CAIX antigen, and it was also demonstrated that CAB - T cells have an in vitro activation ability equivalent to that of the control group BiTA - T, the first - generation CAR, and the second - generation CAR cells.

[0182] In the third experimental group, the inventors detected the in vitro activation ability of the HER2-CAB structure constructed using the trastuzumab-derived scFv sequence. The results are shown in Fig. 11. After co-culturing HER2-CAB-T cells and their control group cells with the HER2-positive SKBR3 cell line or the HER2-negative large cell line for 48 hours respectively, changes in the expression levels of CD137, CD25, CD27, and Ki67 on the surface of the T cell membrane were detected. The results are shown in Fig. 12. When HER2-CAB-T and its control group were co-cultured with large cells respectively, the expression levels of CD137, CD25, CD27, and Ki67 in HER2-CAB-T and its control group immune cells did not change significantly. After co-culturing HER2-CAB-T and its control group with the HER2-positive SKBR3 cells, the expression levels of CD137, CD25, CD27, and Ki67 in T cells modified by HER2-CAB, the first-generation CAR, and the second-generation CAR were significantly upregulated. When T cells modified by tERBB2 and CD3e-BBζ were co-cultured with SKBR3 cells respectively, significant upregulation of CD137, CD25, CD27, and Ki67 was not detected. The above results indicate that HER2-CAB-T cell activation depends on the HER2 antigen, and it is also demonstrated that CAB-T cells have an in vitro activation ability equivalent to that of the control group BiTA-T, the first-generation CAR, and the second-generation CAR cells.

[0183] Example 7 Analysis of unmodified T cells activated by paracrine CAB-T The initial intention in the design of the CAB structure was to achieve the following effects: when encountering tumor cells, CAB-T activates its own anti-tumor activity, while the unmodified T cells surrounding CAB-T are activated by the BiTA drug secreted by CAB-T to achieve paracrine activation. To detect the paracrine activation function of CAB-T in T cells, the inventors conducted verification using a Transwell experiment. Specifically, the experiment was carried out by separating CAB-T and unmodified T cells using a physical barrier of a 0.4 μm Transwell system. The CAB-T cells were placed in the upper chamber, and the unmodified T cells and tumor cells were placed in the lower chamber. The soluble BiTA secreted by CAB-T can freely penetrate into the lower chamber through the 0.4 μm grid and activate the ability of the lower unmodified T cells to recognize and activate tumor cells by recognizing the tumor cells.

[0184] The specific experimental procedure was as follows: 1) 1×10 6 of BiTA-T cells and 1×10 6 of CAB-T were each resuspended in 200 μL of X-VIVO 15 medium and then added to the upper chamber of a 0.4 μm Transwell (Coning, 3413).

[0185] 2) 1×10 6 of unmodified T cells and 1×10 6 of target cells were resuspended in 500 μL of X-VIVO-15 medium and added to the lower chamber of the Transwell.

[0186] 3) The upper chamber was carefully placed into the lower chamber and incubated in an incubator for 48 hours.

[0187] 4) After co-culturing overnight, the culture plates were taken out, and 500 μL of cell supernatant in the lower chamber was taken out. After centrifugation at 300 g for 5 minutes, 150 μL of supernatant / well was taken using a multi-channel pipette and transferred to a new 96-well cell culture plate for ELISA detection of IFN-γ / IL-2 / TNF-α.

[0188] Results: In the second experimental group (Figure 12), both BiTA secreted by CAIX-BiTA-T and BiTA secreted by CAIX-CAB-T were able to activate the ability of the unmodified T cells in the lower part to recognize CAIX-positive MB-231 tumor cells for each pore of the Transwell, showing high release levels of IFN-γ and TNF-α. In addition, the inventors observed no significant change in the IL-2 release level, which was consistent with the results of the study in Example 9.

[0189] From the results shown in the third experimental group (Figure 13), it can be seen that HER2-CAB-T also showed the same paracrine activation on the ability of unmodified T cells to recognize tumor antigens. HER2-CAB-T cells can activate the unmodified T cells in the lower part in the Transwell to recognize HER2-positive SKBR3 tumor cells, while the control tERBB2-T cells cannot. HER2-CAB-T did not assist in the recognition of HER2-negative large cells by unmodified T cells.

[0190] Both the second and third experimental groups showed that the CAB-T structure can activate the ability of unmodified T cells to recognize tumor cells by paracrine BiTA.

[0191] Example 8 Analysis of the Immune Checkpoint Expression Level and Cell Differentiation Phenotype of CAB-T Cells The expression levels of immune checkpoint proteins on immune cells and the differentiation phenotypes of immune cells are closely related to the therapeutic effects of adoptive T cells. Therefore, both lower expression levels of immune checkpoints and a higher proportion of memory T cells predict better clinical response rates. The inventors used flow cytometry to detect the effects of the CAB structure on the immune checkpoint expression levels and cell phenotypes of T cells modified with the CAB structure.

[0192] The specific analysis procedure was as follows: 1) The co-cultured 96-well cell culture plates were centrifuged at 300 g for 5 minutes overnight, 200 μL of FACS buffer was added to each well, and then centrifuged at 300 g for 5 minutes.

[0193] 2) The centrifuged supernatant was removed, the cells were resuspended in 200 μL of FACS buffer, and then centrifuged at 300 g for 5 minutes.

[0194] 3) The antibody was diluted with FACS buffer to prepare an antibody mix (100 μL / well)

[0195]

Table 2

[0196] 4) The centrifuged supernatant was removed, 100 μL of the antibody mix was added to each well, and incubated in the dark at 4 °C for 30 minutes.

[0197] 5) 200 μL of FACS buffer was added to each well, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

[0198] 6) The centrifuged supernatant was removed, and step 2.5 was repeated. 7) 200 μL of Fixation / Permeabilization solution (BD bioscience, 554715) was added and incubated at 4 °C for 20 minutes.

[0199] 8) Centrifuge at 300 g for 5 minutes; remove the centrifuged supernatant, add 200 μL of 1× Perm / Wash buffer (BD bioscience, 554715), resuspend, centrifuge at 400 g for 5 minutes; perform washing twice.

[0200] 9) Dilute the antibody FITC mouse anti-Flag (Biolegend, 637318) 1:1000 with FACS buffer.

[0201] 10) Centrifuge at 400 g for 5 minutes; remove the centrifuged supernatant, and perform washing twice.

[0202] 11) Perform flow cytometry detection using FSC / SSC gating to obtain the desired lymphocyte population, and select the CD3 BV421 + and Flag FITC + cell population to obtain viable CAR-T cells.

[0203] Results: After co-culture with CAIX-positive MB-231 cells, the expression levels of immune checkpoint proteins including LAG-3, PD-1, and TIM-3 on the surface of CAIX-CAB-T cells were lower than those of the second-generation CAR-T modified by CAIX-28ζ. The expression levels of PD-1 and TIM-3 on CAB-T cells were basically the same as those of CAIX-BBζ second-generation CAR-T, and the expression level of LAG-3 in CAB-T cells was slightly lower than that in the second-generation CAR-T modified by CAIX-BBζ, as can be seen from the results of the second experimental group (Figure 14). The lower expression levels of immune checkpoints indicated the clinical superiority of CAB-T cells compared to the second-generation CAR-T.

[0204] In addition, in the second experimental group, the inventors also used CD45RA and CCR7 for the differentiation phenotype analysis of immune cells. The differentiation marker proteins were, respectively, naive T cells (CD45RA + , CCR7 +) Central memory T cells (CD45RA - , CCR7 + ), effector memory T cells (CD45RA - , CCR7 - ), and effector T cells for terminal differentiation (CD45RA + , CCR7 - ). Compared with the second-generation CAR-T, the proportions of cells with the initial differentiated T cell phenotype and central memory T cell phenotype of CAIX-CAB-T were significantly higher than those of the second-generation CAR-T cells, while the proportion of effector memory T cells of the second-generation CAR-T cells was significantly higher than that of the CAR-T cells, as can be seen from the results shown in Figure 14.D. A higher proportion of central memory T cell phenotype predicts better clinical efficacy, thus indicating the superiority of CAB-T in the differentiated state compared with the second-generation CAR-T.

[0205] From the results shown in the third experimental group (Figure 15), it can be seen that the expression status of immune checkpoints and the differentiation results of cell phenotypes for HER2-CAB-T were basically consistent with the analysis results of CAIX-CAB-T. That is, the expression level of immune checkpoints of HER2-CAB-T was lower than that of the second-generation CAR-T modified by CAIX-28ζ and basically equal to or slightly lower than that of the second-generation CAR-T modified by CAIX-BBζ. The differentiation state of HER2-CAB-T also had a higher proportion of central memory T cell phenotype than the second-generation CAR-T.

[0206] Example 9 Antigen-dependent lethal activity of CAB-T Whether CAB-T cells have in vitro lethal activity is an important criterion for judging the potential clinical efficacy of CAB-T. To verify the antitumor activity of CAB-T, the inventors used the LDH method for detection.

[0207] The specific experimental procedure was as follows: 1) An experimental well, an effector cell control well, a target cell control well, a target cell maximum release well, a medium control well, and a volume control well were arranged respectively; the experimental procedure was carried out according to the standard procedure of the CytoTox 96 (registered trademark) Non-Radioactive Cytotoxicity Assay kit (Promega, G1781).

[0208] 2) Different target-effector ratios were set, namely, the number of effector cells: the number of target cells = 0:1, 1:1, 5:1, 10:1, and 20:1.

[0209] 3) The number of cells: 1×10 4 target cells, 50 μL / well. 4) In the experimental wells, 100 μL of cells (50 μL of effector cells + 50 μL of target cells) at different dilution ratios of effector cells: target cells = 0:1, 1:1, 5:1, 10:1, and 20:1 were added to the cell culture plate in triplicate.

[0210] 5) In the effector cell control wells, in duplicate, effector cells: target cells = 0:0, 1:0, 5:0, 10:0, and 20:0.

[0211] 6) In the target cell control wells, 50 μL of target cells at 1×10 4 / well and 50 μL of medium were added.

[0212] 7) In the target cell maximum release wells, 50 μL of target cells at 1×10 4 and 50 μL of medium were added, and 10 μL of lysate was added 1 hour before collecting the samples.

[0213] 8) 100 μL of medium was added to the medium control wells. 9) 100 μL of medium was added to the volume control wells, and 10 μL of lysate was added to the target cell maximum release wells 1 hour before collecting the samples, but 10 μL of lysate was added during that time and the incubation was carried out at 37°C.

[0214] 10) According to the designed layout, the sample was added to the plate and incubated at 37 °C with 5% CO2 for 24 hours, or 36 hours, or 48 hours.

[0215] 11) The assay buffer was taken out from the -20 °C refrigerator and thawed in the refrigerator in the dark at 4 °C. During use, 12 ml of the assay buffer was added to the substrate mix flask and mixed.

[0216] 12) The culture plate was centrifuged at 250 g for 4 minutes, and 50 μL / well of the cell supernatant was transferred to a new ELISA plate.

[0217] 13) 50 μL / well of the substrate mix was added to the new ELISA plate (12 mL of the assay buffer was added to the mix flask and mixed in the dark).

[0218] 14) Incubation was carried out at room temperature for 30 minutes in the dark, and 50 μL / well of the stop solution was added.

[0219] 15) The absorbance was read with a microplate reader at a detection wavelength of OD = 490 nm for 1 hour.

[0220] 16) The cell lethality rate (%) was calculated based on the OD value. Experimental well = Effector - target ratio - Medium control (average) Spontaneous release of target cells = Target cell control - Medium control (average) Spontaneous release of effector cells = Effector cell control - Medium control (average) Maximum release of target cells = Maximum release of target cells (average) - Volume control (average) Cell lethality rate (%) = (Experiment - Spontaneous release of target cells - Spontaneous release of effector cells) / (Maximum release of target cells - Spontaneous release of target cells).

[0221] Results: BiTA secreted by CAB-T can activate CAB-T cells themselves and the non-modified T cells around them in a target antigen-dependent manner, and can kill target antigen-positive tumor cells; on the other hand, CAB-T cells express CD3e-BBζ, so CAB-T may depend on the activation of the endogenous TCR. Furthermore, CD3e-BBζ can enhance the activation level of CAB-T cells, and as a result, CAB-T can be promoted to release more BiTA, and these effects reinforce each other. Therefore, theoretically, CAB-T should have a stronger lethal effect on tumor cells than BiTA-T.

[0222] In the first experimental group, CAIX + To detect the lethal effect of CAIX-CAB-T cells on HEK 293T target cells, the inventors selected CAIX-CAB-T cells as the effector cells of the experimental group for lethal effect detection. The effector cells were co-cultured with the target cells at effector-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1 for 24 and 48 hours respectively, and the supernatant was collected to determine the ability of T cells to kill target cells at different effector-target ratios. The first CAIX-CAB-T cells and their control group T cells had CAIX - No lethal effect on HEK293T, while the first BiTA-T cells, the first CAIX-CAB-T, CAIX-TRuC-T, and the mixed T cells of CD3e-BBζ-T and the first BiTA-T showed different degrees of lethal ability against CAIX + HEK 293T cells, and it can be seen from the results of Figure 16 that this lethal ability increased with the increase of the effector-target ratio. Furthermore, the lethality of target cells by CAIX-CAB-T and its control group T cells became more significant with the extension of the co-culture time, and the lethal effect of 48-hour co-culture was significantly stronger than that of 24-hour co-culture. CD3e-BBζ-T and the non-modified T cell control group had CAIX +In addition, the primary CAIX-CAB-T, CAIX-TRuC-T, and CD3e-BBζ-T / primary BiTA-T T cells showed no lethal effect on HEK 293T cells. + The CAIX-CAB-T cells had comparable lethality against HEK 293T cells and were more potent than the first BiTA-T. Therefore, we conclude that the lethality of CAIX-CAB-T cells against tumor cells depends on the expression of their target antigen and is comparable to that of the control CAIX-TRuC-T cells. Additionally, BiTA-T and CD3e-BBζ-T cells exhibited a synergistic effect on target cell killing.

[0223] The method for the second experimental group was the same as that for the first experimental group. + MB-231 or CAIX - The killing ability of CAIX-CAB-T and its control cells against MB-231 tumor cells was detected. Effector cells were co-cultured with target cells at effector-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1 for 36 hours, and the supernatants were collected to determine the ability of T cells to kill target cells at different effector-target ratios. CAIX-CAB-T and its control cells were co-cultured with CAIX. - The lack of lethal effect on MB-231 control tumor cells and the fact that CAIX-CAB-T, as well as first- and second-generation CAR-T cells targeting CAIX, were not associated with lethal effects on CAIX. + The results shown in Figure 17 show that CAIX showed comparable killing ability against MB-231 cells. In addition, tERBB2-T and CD3e-BBζ-T control cells showed similar killing ability against MB-231 cells. + CAIX-CAB-T lacked lethality against MB-231 cells. CAIX-CAB-T was found to have comparable lethality against tumor cells to first- and second-generation CAR-Ts, and this lethality was dependent on the target antigen. Note that CAIX-CAB-T and CAIX-BiTA-T did not demonstrate differences in lethality against target cells in this experimental group due to higher transduction levels or different donor cell sources or the like.

[0224] The third experimental group was the same as the first and second experimental groups. The inventors detected the ability of HER2-CAB-T and its control cells to kill HER2-positive tumor cells SKBR3 or HER2-negative tumor cells large. Effector cells were co-cultured with target cells for 36 hours at effector-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1, respectively, and the supernatant was collected to determine the ability of T cells to kill target cells at different effector-target ratios. It can be seen from the results shown in Figure 18 that HER2-CAB-T and its control cells had no lethal effect on HER2-negative large cells, and HER2-CAB-T and first-generation and second-generation CAR-T cells showed equivalent lethal ability against SKBR3. In addition, tERBB2-T and CD3e-BBζ-T control cells had no lethal ability against SKBR3 cells. It was revealed that HER2-CAB-T had an equivalent lethal ability to first-generation and second-generation CAR-T against tumor cells, and this lethal ability was target antigen-dependent.

[0225] Example 10 Comparison of in vitro activities of different CAB structures having BiTA and second-generation CAR Since the expression levels of CD3e-BBζ and BiTA in cells are different, the inventors attempted to analyze the differences between different structures of CAB, BiTA, and second-generation CAR. The inventors first designed a group of CAB and its control structures, including tERBB2, HER2-BiTA, HER2-CAB, HER2-CAB R (DNA sequence number SEQ 67, AA sequence number SEQ 68) and HER2-BBζ, as shown in Figure 19. Lentiviruses were packaged using vectors having the above structures according to the method described in Example 2 and T cells were infected according to the method described in Example 3. The positive frequency of infected cells was detected according to the method described in Example 4, and the detection results are shown in Figure 20. The positive frequencies of the modified T cells were basically equal.

[0226] According to the method described in Example 5, to identify the in vitro activation ability of the modified T cells, the modified T cells were co-cultured with HER2-negative large cells and HER2-positive SKBR3 cells respectively, and then the release levels of cytokines IL-2 and IFNγ were detected. The results are shown in Figure 21. HER2-CAB-T and HER2-CAB R The cytokine release levels after activation of -T cells were basically equal.

[0227] To identify the difference in the in vitro killing ability of the modified T cells, the inventors detected the in vitro killing ability of each of the modified T cells against large and SKBR3 cells according to the method described in Example 9. Compared with the HER2-CAB structure, HER2-CAB R structure has the BiTA structure in the front and the CD3e-BBζ structure in the back. Therefore, theoretically, HER2-CAB R -T can secrete a higher level of BiTA. The inventors speculated that HER2-CAB R -T has better in vitro killing ability than HER2-CAB-T cells. As shown in Figure 22, the results are consistent with the inventors' hypothesis, that is, HER2-CAB R -T showed better in vitro killing ability than HER2-CAB-T cells.

[0228] Example 11 In vivo efficacy of CAIX CAB-T The anti-tumor activity of CAB-T in a mouse tumor model is an important criterion for judging the potential clinical efficacy of CAB-T. To verify the anti-tumor activity of CAB-T in a mouse tumor model, the following verification experiments were conducted.

[0229] The specific experimental procedure was as follows. CAIX + MDA-MB-231 cell expansion culture and inoculation 1) Sufficient CAIX +MDA-MB-231 cells were cultured and expanded in vitro. After trypsin digestion, the cells were collected, washed three times with PBS, counted, and resuspended at a cell density of 15×10 6 cells / ml using 80% RPMI-1640 basal medium containing 20% Matrigel. The cells were placed in 50 ml centrifuge tubes, the openings of which were covered and sealed with sealing film, and the centrifuge tubes were transferred to an SPF-grade animal room via a transfer window.

[0230] 2) Sixty-eight female 8-week-old NCG severely immunodeficient mice, which had been purchased in advance, were adaptively fed for one week, and then the hair on the right abdomen of each mouse was removed with a razor. CAIX 6 MDA-MB-231 cells with a density of 15×10 + cells / ml were dissociated, carefully mixed with a 1 ml pipette, and 0.2 ml of the cells were subcutaneously inoculated into the right abdomen of each NCG mouse using a 1 ml syringe, i.e., 3×10 6 CAIX + MDA-MB-231 cells were inoculated. The cells were observed daily for subcutaneous tumor formation in the NCG mice, and each NCG mouse was numbered using numbered ear tags 6 days after inoculation.

[0231] CAIX + Grouping, administration, and measurement of CAIX MDA-MB-231 tumor-bearing mice

[0232] ​4) For each group, grouping was performed according to the following dosing scheme, and the corresponding reagent or cells were injected. In the hCAIX-BiTA (DNA sequence number SEQ 71, AA sequence number SEQ 72), hCAIX-CAB (DNA sequence number SEQ 73, AA sequence number SEQ 74), and hCAIX-BBζ (DNA sequence number SEQ 75, AA sequence number SEQ 76) constructs for modifying T cells, all antibody sequences for recognizing CAIX are derived from the humanized sequence of the VHH amino acid sequence, sequence number 18.

[0233] Dosing scheme for each group

[0234]

Table 3

[0235] 5) The tumor volume and body weight of the mice were measured twice a week. The body weight and tumor volume of the mice were measured for the last 37 days after inoculation with tumor cells. After euthanizing the mice, the tumors of each mouse were excised, the tumor weight was measured, and photographs of the tumors were taken.

[0236] Results and discussion The tumor growth curves, tumor photographs, and tumor weights of each group of mice are shown in the figures. The tumors of the mice in the PBS group and the NT group increased rapidly with the extension of the inoculation time. This indicates that the establishment of the CDX transplantation model was successful in this experiment. Compared with the PBS group and the NT group, the hCAIX-BiTA-T cell group and the hCAIX-BBζ-T cell group showed an inhibitory effect on tumor growth only at 2.5×10 6 / mouse, while the hCAIX-CAB-T cell group had a certain effect in the three dose groups, and these effects were dose-dependent. All tumors regressed in the 2.5×10 6 / mouse group, and in the 0.75×10 6In 5 out of the mouse group, there was tumor regression, and the hCAIX-CAB-T cell group was significantly superior to the hCAIX-BiTA-T cell group and the hCAIX-BBζ-T cell group at the same dose. The trends of the tumor growth curves, tumor photographs, and tumor weight results of each group were consistent (Figures 23A, C, and D). There was no significant increase in the body weight of the mice in each group throughout the experimental period (Figure 23B). This indicates the safety of each test sample.

[0237] Example 12 In Vivo Efficacy of HER2 CAB-T The antitumor activity of HER2 CAB-T was verified by the following experiments in an M-NSG immunodeficient mouse tumor model.

[0238] Expansion Culture and Inoculation of NCI-N87 Cells 1) NCI-N87 cells were cultured and expanded in vitro. After trypsin digestion, the cells were collected, washed 3 times with PBS, counted, and the cell density was adjusted to 10×10 6 cells / ml using 80% RPMI-1640 basal medium containing 20% Matrigel. The cells were placed in a 50 ml centrifuge tube, the opening of the centrifuge tube was covered and sealed with a sealing film, and the centrifuge tube was transferred to an SPF-grade animal room via a transfer window.

[0239] 2) Thirty-two 8-week-old female NSG severely immunodeficient mice were adaptively fed, and the hair on the right abdomen of each mouse was removed with a razor. NCI-N87 cells with a density of 10×10 6 cells / ml were dissociated, carefully mixed with a 1 ml pipette, and 0.2 ml of the cells were subcutaneously inoculated into the right abdomen of each NSG mouse with a 1 ml syringe, that is, 3×10 6 NCI-N87 cells were inoculated into each NSG mouse. The cells were observed daily for subcutaneous tumor formation in NSG mice, and each NSG mouse was numbered using numbered ear tags 6 days after inoculation.

[0240] Grouping, Administration, and Measurement of NCI-N87 Tumor-Bearing Mice 3) Six days after inoculation, the widest axis W and the longest axis L of the subcutaneous tumor in the right abdomen of each NSG mouse were measured using calipers, and the body weight of each mouse was weighed using an electronic balance. The subcutaneous tumor volume in the right abdomen of each NCG mouse was calculated according to the tumor volume formula: T = 1 / 2 × W × W × L. Mice with tumors that were either too large or too small were excluded, and the NSG mice were evenly divided into four groups of six mice each according to the average tumor volume.

[0241] 4) For each group, grouping was performed according to the following dosing scheme, and the corresponding reagent or cells were injected. NT (DNA sequence number SEQ 33, AA sequence number SEQ 34), HER2 CAB R -T (DNA sequence number SEQ 67, AA sequence number SEQ 68), HER2 CAB-T (DNA sequence number SEQ 49, AA sequence number SEQ 50), and in the HER2 CAR-T construct, all scFv antibody sequences for recognizing HER2 are derived from the trastuzumab amino acid sequence, sequence number 66.

[0242] Dosing scheme for each group

[0243]

Table 4

[0244] 5) The tumor volume and body weight of the mice were measured twice a week. For the last 48 days after inoculation with tumor cells, the body weight and tumor volume of the mice were measured. After euthanizing the mice, the tumors of each mouse were excised, the tumor weight was weighed, and a photo of the tumor was taken.

[0245] Results and discussion Tumor growth curves and tumor photos for each group of mice are shown in the figure. Tumors in the NT group of mice increased rapidly with the extension of the inoculation time. This indicates that the establishment of the CDX transplantation model was successful in this experiment. Compared with the NT group, all of the other three modified T cell treatment groups showed a certain degree of tumor growth inhibition. In contrast to the mild tumor growth inhibition and two tumor-free mice in the HER2 CAR-T treatment group, both HER2-CAB R -T and HER2-CAB-T showed much better tumor growth inhibition and had five and two tumor-free mice, respectively.

[0246] The trends of the results of the tumor growth curves, tumor photos and tumor weights of each group were consistent (Figure 24A, C and D). There was no significant increase in the body weight of the mice in each group throughout the experimental period. This indicates that each test sample had good safety.

[0247] Example 13 Dasatinib acts as a safety switch and inhibits cytokine release of CAB-T In the case of live cell therapy, CAB-T may cause cytokine release syndrome (CRS) and on-target, off-tumor toxicity, etc. The clinical management of CRS includes anti-IL-6R agonist tocilizumab and steroid treatment. Here, the inventors developed a method to manage potential toxicity using dasatinib.

[0248] Dasatinib was developed as an inhibitor of the BCR-ABL fusion protein and its clinical use for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia has been approved. In addition, it has been reported that dasatinib can block lymphocyte-specific protein tyrosine kinase (LCK), thereby inhibiting the phosphorylation of CD3ζ and ZAP70 and disrupting signal transduction in CAR and TCR signal transduction. The following examples demonstrate that dasatinib can dramatically inhibit the cytokine release of CAB-T.

[0249] 1×10 5effector cells (CAIX CAB-T or HER2 CAB-T) and 1×10 5 target cells (CAIX + MDA-MB231 or SKBR3) were seeded at 200 μL / well in a 96-well cell culture plate together with gradient concentrations of dasatinib (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM), co-cultured overnight, then the plate was centrifuged at 300 g for 5 minutes, and then 150 μL of the supernatant / well was transferred to a new 96-well cell culture plate using a multi-channel pipette, and cytokine detection was performed as shown in Example 5.

[0250] Results and Discussion As shown in Figures 25A, 25B and Figures 26A, 26B, dasatinib effectively inhibited IFNγ and IL-2 secretion in both activated CAIX CAB-T and HER2 CAB-T at a low dose of approximately 25 nM. This result implies that dasatinib could be used as a safety switch to control potential CRS syndrome and on-target, off-tumor toxicity of CAB-T in clinical treatment.

[0251] Example 14 Dasatinib acts as a safety switch and inhibits the lethal activity of CAB-T As described in Example 13, T cell therapy has potential clinically on-target, off-tumor toxicity problems. To manage the potential toxicity of CAB-T, dasatinib was used to evaluate the inhibition of the lethal activity of CAB-T.

[0252] The inventors used the LDH cytotoxicity assay method to detect the lethal inhibitory ability of dasatinib. The detailed protocol was described in Example 9. Briefly, 5×10 4 effector cells (CAIX CAB-T or HER2 CAB-T) and 1×10 4 target cells (CAIX +MDA-MB231 or SKBR3) were co-cultured with gradient concentrations of dasatinib (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM), and the cytotoxicity of CAB-T against its target cells was determined at 2-hour intervals over a 12-hour period [effector-to-target cell (E:T) ratio, 5:1].

[0253] Results and Discussion As shown in Figures 25C and 26C, dasatinib effectively inhibited the cytotoxicity of CAIX CAB-T and HER2 CAB-T against their target cells at a low dose of approximately 50 nM. This result implies that dasatinib could be used as a safety switch to control the potential off-target, off-tumor toxicity of CAB-T in clinical treatment.

[0254] Summary and Discussion: CAB-T technology utilizes BiTA secreted by itself to simultaneously recognize chimeric CD3 and tumor antigen and endogenous CD3 in T cells, and then induces the activation of endogenous TCR and chimeric CD3 that depends on tumor antigens. The activation of both the endogenous TCR complex and chimeric CD3 depends on the expression and secretion levels of BiTA by CAB-T cells. Therefore, BiTA can induce CAB-T cells through autocrine and activate non-modified T cells in the tumor microenvironment through paracrine. On the other hand, CAB-T cells can mobilize the activation of more non-modified T cells and the anti-tumor effect in the tumor tissue by releasing a high level of BiTA into the tumor tissue after activation in the tumor tissue.

[0255] Therefore, compared with TRuC-T and TAC-T, CAB-T not only has the advantage of activating the endogenous TCR signal, but also mobilizes the anti-tumor activity of infiltrating T cells into the tumor tissue, and theoretically has better therapeutic potential for solid tumors. In addition, different from BiTE drugs, the BiTA drug continuously secreted by CAB-T cells solves the clinical application problem of the short half-life of a single BiTE drug. In addition, BiTA targeting the target antigen will exert the greatest effect in the tumor microenvironment reached by CAB-T cells and will not be concentrated at high concentrations in non-tumor tissue sites. Therefore, BiTA has better safety and greater potential for clinical application compared with the systemic administration of a single BiTE drug.

[0256] The mechanism of action and clinical application potential of CAB-T are described below. 1) In tumor tissues, BiTA, which is underexpressed in CAB-T cells, can bind to its own endogenous TCR complex or CD3e-BBζ in an autocrine manner (Figure 27 B), thereby stimulating CAB-T to release more BiTA and realizing a local activation loop (Figure 27 C). As a result, CAB-T can reach the maximum activation level at the local tumor site, and thus exert its maximum anti-tumor effect, realizing the same safety and efficacy as local administration of drugs to the tumor site. CD3e-BBζ forms a heterodimer with the endogenous CDδ or CD3γ chain on the T cell membrane, and thus is predicted to further enhance CD3-based signal transduction.

[0257] 2) The binding and activation of chimeric CD3e in CAB-T by autocrine BiTA (Figure 27 A) endows CAB-T with enhanced proliferation and activation abilities that are more sensitive than unmodified T cells, and thus further enhances the anti-tumor activity of CAB-T.

[0258] 3) Autocrine and paracrine of BiTA on CAB-T (Figure 27A, B, and C) and unmodified T cells (Figure 27 D) activation solves the problem that CAR-T cells cannot activate the endogenous TCR signal, thus providing the possibility of treating solid tumors with CAB-T cells.

[0259] 4) CAB-T can be used as a drug synthesis factory for BiTA, thereby solving the problem of the in vivo half-life of BiTA. The activation of CAB-T depends on the release level of BiTA, and these two are interdependent and cooperate with each other to jointly determine the safety and efficacy of CAB-T for clinical applications.

[0260] An overview diagram of the mechanism of action of CAB-T is shown in Figure 27. All documents mentioned in this disclosure are hereby incorporated by reference as if each document was individually listed as a reference. In addition, those skilled in the art should understand that after reading the above teachings of this disclosure, various modifications or changes of this disclosure can be implemented, and these equivalent forms are also included within the scope defined by the claims appended hereto.

Claims

1. A nucleic acid molecule encoding a chimeric CD3 fusion protein and a bispecific T cell activation element, wherein the chimeric CD3 fusion protein comprises one or more polypeptides (EC) recognizable by an anti-CD3 antibody, a transmembrane domain (TM), and a CD3 signal activation domain, where EC is a polypeptide from or derived from CD3e, the bispecific T cell activation element is a fusion protein comprising one or more tumor antigen recognition regions and one or more CD3 antigen-binding antibody fragments.

2. The nucleic acid molecule according to claim 1, wherein the chimeric CD3 fusion protein comprises one or more of the following domains: a hinge or linker region, and a co-stimulatory domain.

3. The fusion protein has a structure represented by the following formula I: L-EC-H-TM-C-CD3ζ (I) (wherein, L is absent or is a signal peptide sequence; EC is a polypeptide binding domain recognizable by an anti-CD3 antibody, which binds to the anti-CD3 antibody; H is absent or is a linker or hinge region; TM is a transmembrane domain; C is absent or is a co-stimulatory signaling molecule; CD3ζ is absent or is a cytoplasmic signaling sequence derived from CD3ζ; each “-” is independently a linker peptide or a peptide bond) The nucleic acid molecule according to claim 1.

4. The bispecific T cell activation element has a structure represented by the following formula II: L’-T1-B1-B2-T2 (II) (wherein, L’ is absent or is a signal peptide sequence; T1 is absent or is a tag element; B1 is a tumor antigen recognition region or a CD3 antigen-binding antibody fragment; B2 is a CD3 antigen-binding antibody fragment or a tumor antigen recognition region; T2 is absent or is a tag element; each “-” is independently a linker peptide or a peptide bond) The nucleic acid molecule according to claim 1.

5. The nucleic acid molecule according to claim 1, wherein EC comprises amino acid positions 1 to 104 of the CD3e protein having the amino acid sequence represented by SEQ ID NO: 4 and / or the nucleotide sequence represented by SEQ ID NO:

3.

6. The nucleic acid molecule according to claim 3, wherein when C is present, it is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

7. The nucleic acid molecule according to claim 1, wherein when C is present, it comprises a co-stimulatory signal molecule derived from 4-1BB having the amino acid sequence represented by SEQ ID NO: 10 and / or the nucleotide sequence represented by SEQ ID NO: 9, and / or a co-stimulatory signal molecule derived from CD28 having the amino acid sequence represented by SEQ ID NO: 62 and / or the nucleotide sequence represented by SEQ ID NO:

61.

8. The nucleic acid molecule according to claim 3, wherein when CD3ζ is present, it is a cytoplasmic signaling sequence having the amino acid sequence represented by SEQ ID NO: 12 and / or the nucleotide sequence represented by SEQ ID NO:

11.

9. The nucleic acid molecule according to claim 4, wherein B1 is a tumor antigen recognition region and B2 is a CD3 antigen recognition region.

10. The nucleic acid molecule according to claim 4, wherein the tumor antigen recognition region comprises a receptor or ligand binding domain, an antibody fragment, and / or a T cell receptor (TCR) sequence.

11. The nucleic acid molecule according to claim 4, wherein the tumor antigen recognition region targets tumor antigens CAIX and / or HER2.

12. The nucleic acid molecule according to claim 4, wherein the tumor antigen recognition region is an antibody fragment having the amino acid sequence represented by SEQ ID NO: 18 and / or the nucleotide sequence represented by SEQ ID NO:

17.

13. The nucleic acid molecule according to claim 4, wherein the tumor antigen recognition region targets HER2 and is an antibody fragment having the amino acid sequence represented by SEQ ID NO: 66 and / or the nucleotide sequence represented by SEQ ID NO:

65.

14. The nucleic acid molecule according to claim 4, wherein the CD3 antigen-binding antibody fragment targeting CD3 comprises a single domain antibody sequence (VHH), a single-chain antibody variable region sequence (scFv), and / or an antigen-binding fragment (Fab).

15. The nucleic acid molecule according to claim 4, wherein the CD3 antigen-binding antibody fragment is derived from an anti-CD3 Ab clone.

16. The nucleic acid molecule according to claim 1, comprising the sequence represented by SEQ ID NO:

73.

17. The nucleic acid molecule according to claim 1, comprising the sequence represented by SEQ ID NO:

67.

18. A vector comprising the nucleic acid molecule according to any one of claims 1 to 17.

19. A genetically modified immune cell that expresses a protein encoded by the nucleic acid molecule according to any one of claims 1 to 17.

20. The genetically modified immune cell according to claim 19, which is a T cell.

21. An unnatural T cell population comprising the genetically modified immune cell according to claim 20, wherein the T cell is present in the T cell population at a ratio C1 of 10% or higher based on the total number of T cells in the T cell population.

22. A composition comprising (a) the genetically modified immune cell according to claim 20 and / or the T cell population according to claim 21, and (b) a pharmaceutically acceptable carrier, diluent and / or excipient, wherein the immune cell is a T cell.

23. Use of the genetically modified immune cell according to claim 20 and / or the T cell population according to claim 21 in the preparation of a medicament for the prevention and / or treatment of cancer or tumor, wherein the immune cell is a T cell.

24. The use according to claim 23, wherein the tumor is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

25. The use according to claim 23, wherein the medicament is used in combination with dasatinib.

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