Design of a bispecific t cell activator to activate t cells and uses thereof

Abstract

This invention provides an immunotherapy regimen for inhibiting tumors, especially solid tumors—combining T cells expressing chimeric CD3e fusion protein with BiTA. The chimeric CD3e fusion protein and BiTA bind to each other, activating T cells and targeting tumor cells. This invention also provides a CAB structure and CAB-edited T cells (CAB-T). BiTA secreted by CAB-T cells in tumor tissue can simultaneously activate CAB-T cells and the endogenous TCRs of non-edited T cells, exerting both CAB-T's own anti-tumor effects and mobilizing the anti-tumor effects of unedited T cells. The chimeric CD3e expressed by CAB-T and BiTA synergistically exert anti-tumor effects: the activation of chimeric CD3e depends on BiTA secreted by CAB-T, and the secretion of BiTA further stimulates CAB-T to release more BiTA by activating chimeric CD3e; the small amount of BiTA released by CAB-T in tumor tissue can stimulate CAB-T to release more BiTA by activating chimeric CD3e, achieving tumor-preferred immune cell activation and anti-tumor effects, ensuring the safety advantage of CAB-T in clinical applications.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy. Specifically, this invention relates to the design and application of a bispecific T cell activator for activating T cells. Background Technology

[0002] In recent years, immunotherapy has achieved unprecedented success in achieving complete remission rates for hematological malignancies. In 2017, two CD19-targeted CAR-T products were successfully launched and approved for the treatment of acute leukemia and non-Hodgkin's lymphoma in children and adolescents, respectively.

[0003] However, two major challenges remain in using immunotherapy to treat solid tumors: First, the severe and even fatal clinical side effects associated with CAR-T therapy remain a significant risk to its clinical application. These side effects mainly include cytokine release syndrome (CRS), macrophage activation syndrome, hemophagocytic lymphohistiocytoma, and neurotoxicity. Second, CAR-T therapy has not yet demonstrated significant clinical efficacy in the treatment of solid tumors.

[0004] Therefore, there is an urgent need in this field to develop a treatment regimen for inhibiting solid tumors that has good clinical efficacy and low clinical side effects. Summary of the Invention

[0005] The purpose of this invention is to provide a treatment plan for inhibiting solid tumors with good clinical efficacy and low clinical side effects.

[0006] The first aspect of this invention provides a fusion protein, characterized in that the structure of the fusion protein is shown in Formula I:

[0007] L-EC-H-TM-C-CD3ζ (I)

[0008] In the formula,

[0009] L represents the absence of a signal peptide sequence;

[0010] EC is a polypeptide binding domain, which can be recognized by CD3 antibodies and bind to CD3 antibodies.

[0011] The peptide-binding domain is also called the recognition-binding domain of the CD3 antibody;

[0012] H represents the area without hinges;

[0013] TM represents a transmembrane domain;

[0014] C represents a non- or co-stimulatory signaling molecule;

[0015] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

[0016] Each "-" independently represents a linking peptide or peptide bond.

[0017] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, GM-CSF, CD4, CD137, or a combination thereof.

[0018] In another preferred embodiment, the polypeptide binding domain is derived from or derived from the CD3e protein.

[0019] In another preferred embodiment, the polypeptide binding domain is the extracellular region of CD3e or other amino acid sequences that can be recognized by CD3 antibodies.

[0020] In another preferred embodiment, the CD3 antibody is selected from the group consisting of scFV (single-chain antibody), nanobody, double-chain antibody, or variants thereof, or combinations thereof.

[0021] In another preferred embodiment, the CD3 antibody comprises the CD3 antibody segment of a BiTA molecule.

[0022] In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to the CD3 antibody.

[0023] In another preferred embodiment, the EC is the first to the fourth position of the wild-type or mutant CD3e protein, and its amino acid sequence is shown in SEQ ID NO.:1.

[0024] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0025] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0026] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: 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 combinations thereof.

[0027] In another preferred embodiment, C includes co-stimulatory signaling molecules derived from 4-1BB and / or co-stimulatory signaling molecules derived from CD28.

[0028] A second aspect of the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes a fusion protein as described in the first aspect.

[0029] A third aspect of the present invention provides a carrier, characterized in that the carrier contains nucleic acid molecules as described in the second aspect.

[0030] In another preferred embodiment, the vector includes a lentiviral vector, adenovirus, or retrovirus.

[0031] A fourth aspect of the present invention provides a genetically engineered T cell, characterized in that the T cell expresses the chimeric fusion protein as described in the first aspect.

[0032] In another preferred embodiment, the T cells are derived from humans or non-human mammals.

[0033] In another preferred embodiment, the T cells also include other chimeric antigens.

[0034] A fifth aspect of the present invention provides a genetically engineered T cell, characterized in that the T cell expresses (a) a bispecific T cell activation element BiTA based on anti-CD3, and (b) a fusion protein as described in the first aspect.

[0035] In another preferred embodiment, the structure of (a) the anti-CD3-based bispecific T cell activation element BiTA is shown in Formula II:

[0036] L'-T1-B1-B2-T2 (Ⅱ)

[0037] In the formula,

[0038] L' represents the absence of a signal peptide sequence;

[0039] T1 is an element without a label;

[0040] B1 is the tumor antigen recognition region;

[0041] B2 is the CD3 antigen recognition region;

[0042] T2 is an element without a label;

[0043] Each "-" independently represents a linking peptide or peptide bond.

[0044] In another preferred embodiment, L' is a signal peptide of a protein selected from the group consisting of CD8, GM-CSFR, CD4, CD137, or a combination thereof.

[0045] In another preferred embodiment, the tag element includes a tag protein, a fluorescein-tagged protein, or an enzyme-tagged protein.

[0046] In another preferred embodiment, the tag protein includes FLAG protein and His protein.

[0047] In another preferred embodiment, the tumor antigen recognition region B1 comprises a single-domain antibody sequence (VHH) and / or a single-chain antibody variable region sequence (scFv).

[0048] In another preferred embodiment, the tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, 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 α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, liver glycoprotein B2, IGF-I receptor, CAIX, LMP2, gp100. bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, 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, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin8, 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 combinations thereof.

[0049] In another preferred embodiment, the tumor antigen recognition region B1 targets CAIX and / or HER2.

[0050] In another preferred embodiment, the tumor antigen recognition region B1 is a nanobody targeting CAIX.

[0051] In another preferred embodiment, the tumor antigen recognition region B1 is a single-chain antibody targeting HER2.

[0052] In another preferred embodiment, the CD3 antigen recognition region B2 is a single-domain antibody sequence (VHH) targeting CD3, and / or a single-chain antibody variable region sequence (scFv).

[0053] In another preferred embodiment, the BiTA is a secretory BiTA.

[0054] In another preferred embodiment, the secretory BiTA may be autocrine and / or paracrine.

[0055] In another preferred embodiment, the BiTA may be combined with a chimeric CD3e.

[0056] In another preferred embodiment, the BiTA can bind to the T cell receptor TCR.

[0057] In another preferred embodiment, the TCR is derived from T cells as described in the fifth aspect, and / or unengineered T cells.

[0058] A sixth aspect of the present invention provides a composition comprising the fusion protein as described in the first aspect and BiTA.

[0059] In another preferred embodiment, the fusion protein in the composition is located in the extracellular region of the T cell membrane as described in the first aspect.

[0060] In another preferred embodiment, the BiTA in the composition is autocrine, paracrine, or exogenous BiTA.

[0061] In another preferred embodiment, the composition is expressed as a fusion expression of a protein of structural formula I and structural formula II and a 2A protein, the structural formula of which is: I-2A-II or II-2A-I, wherein the 2A sequence includes one or a combination of T2A, P2A, F2A or E2A.

[0062] In another preferred embodiment, the composition is expressed as a combination of a fusion protein of structural formula I and structural formula II and an IRES sequence, the structural formula of which is: I-IRES-II or II-IRES-I, wherein IRES is a ribosome entry site nucleotide sequence.

[0063] In another preferred embodiment, the IRES functions to initiate amino acid translation of downstream genes.

[0064] The seventh aspect of the present invention provides a genetically engineered T cell, characterized in that the T cell expresses the composition described in the fifth aspect.

[0065] The eighth aspect of the present invention provides a non-naturally occurring T cell population, wherein the proportion of the T cells described in the fourth and fifth aspects in the T cell population is C1≥10%, based on the total number of T cells in the T cell population.

[0066] In another preferred embodiment, C1 ≥ 10%, more preferably C1 ≥ 20%, and even more preferably C1 ≥ 30%.

[0067] In another preferred embodiment, the T cell population is described.

[0068] In another preferred embodiment, the T cell population also contains BiTA and / or BiTA-secreting T cells C2.

[0069] The ninth aspect of the present invention provides a cell preparation comprising (a) T cells as described in the fourth aspect, T cells as described in the fifth aspect, T cells as described in the seventh aspect, and / or a population of T cells as described in the eighth aspect, and (b) a pharmaceutically acceptable carrier, diluent, or excipient.

[0070] The tenth aspect of the present invention provides the use of T cells as described in the fourth aspect, T cells as described in the fifth aspect, T cells as described in the seventh aspect, and / or T cell populations as described in the eighth aspect, and / or cell preparations as described in the ninth aspect, for the preparation of medicaments for the prevention and / or treatment of cancer or tumors.

[0071] In another preferred embodiment, the tumor is selected from the group consisting of hematologic malignancies, solid tumors, or combinations thereof.

[0072] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0073] In another preferred embodiment, the solid tumor is selected from the group consisting of: 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 carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or combinations thereof.

[0074] In another preferred embodiment, the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, liver cancer, endometrial cancer, or combinations thereof.

[0075] The eleventh aspect of the present invention provides a method for treating a disease, comprising: administering to a subject requiring treatment an appropriate amount of T cells as described in the fourth aspect, T cells as described in the fifth aspect, T cells as described in the seventh aspect, and / or a population of T cells as described in the eighth aspect, and / or a cell preparation as described in the ninth aspect.

[0076] In another preferred embodiment, the disease is cancer or a tumor.

[0077] In another preferred embodiment, the method further includes administering appropriate amounts of cytokines or pharmaceutical compounds and combinations thereof that stimulate cell secretion to enhance the responsiveness of immune cells.

[0078] In another preferred embodiment, the immune cells include T cells as described in the fourth aspect, T cells as described in the fifth aspect, T cells as described in the seventh aspect, and / or T cell populations as described in the eighth aspect, and / or cell preparations as described in the ninth aspect, as well as endogenous T cells, NK cells, macrophages, and B cells.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0080] Figure 1 The structures of the first experimental group CAB-T and its control group are shown.

[0081] Figure 2 The structures of the second experimental group CAB-T and its control group are shown.

[0082] Figure 3 The structures of the third experimental group CAB-T and its control group are shown.

[0083] Figure 4 The results of the first group of structural editing T cell positivity rate detection are shown.

[0084] Figure 5 The results of the second set of structural editing T cell positivity rate assays are shown.

[0085] Figure 6 The results of the third group of structural editing T cell positivity rate detection are shown.

[0086] Figure 7The CAIX-CAB-T cytokine release assay (Group 1 experiment) is shown.

[0087] Figure 8 The CAIX-CAB-T cytokine release assay (second group of experiments) is shown.

[0088] Figure 9 The detection of CAIX-CAB-T cell activation levels is shown (Group 1 experiment).

[0089] Figure 10 The detection of CAIX-CAB-T cell activation levels is shown (second group of experiments).

[0090] Figure 11 The assay showed the activation level of HER2-CAB-T cells (third group of experiments).

[0091] Figure 12 The CAIX-CAB-T paracrine activated T cell level assay is shown (Group 2).

[0092] Figure 13 The level of HER2-CAB-T paracrine activated T cells was detected (Group 3).

[0093] Figure 14 The expression levels of immune checkpoints and cell differentiation phenotypes in CAIX-CAB-T cells and their control cells are shown in the second group.

[0094] Figure 15 The expression levels of immune checkpoints in HER2-CAB-T cells and their control cells, as well as cell differentiation phenotype analysis, are shown (third group of experiments).

[0095] Figure 16 The tumor-killing ability mediated by CAIX-CAB-T cells and their control group was demonstrated (Group 1 experiment).

[0096] Figure 17 The tumor-killing ability mediated by CAIX-CAB-T cells and their control group was demonstrated (second group experiment).

[0097] Figure 18 The tumor-killing ability mediated by HER2-CAB-T cells and their control group was demonstrated (third group experiment).

[0098] Figure 19 A schematic diagram of the CAB-T mechanism of action is shown. Detailed Implementation

[0099] Through extensive and in-depth research, the inventors have provided an immunotherapy regimen for inhibiting tumors, especially solid tumors—combining T cells expressing a chimeric CD3e fusion protein with BiTA. The chimeric CD3e fusion protein and BiTA bind to each other, activating T cells and targeting tumor cells. This invention also provides a CAB structure and CAB-edited T cells (CAB-T), in which the edited T cells express chimeric CD3e and a bispecific T cell activator based on a CD3 antibody. The BiTA secreted by CAB-T cells can simultaneously activate CAB-T cells and the endogenous TCRs of non-edited T cells in tumor tissue, exerting both the CAB-T cells' own anti-tumor effects and mobilizing the anti-tumor effects of unedited T cells, thus ensuring the effectiveness of CAB-T in clinical applications. The chimeric CD3e expressed by CAB-T cells synergistically exerts an anti-tumor effect with BiTA: the activation of chimeric CD3e depends on BiTA secreted by CAB-T cells, and the secretion of BiTA further stimulates CAB-T cells to release more BiTA by activating chimeric CD3e; the small amount of BiTA released by CAB-T cells in tumor tissue can stimulate CAB-T cells to release more BiTA by activating chimeric CD3e, thereby achieving tumor-preferred immune cell activation and anti-tumor effects, ensuring the safety advantage of CAB-T cells in clinical applications. Based on this, the inventors completed this invention.

[0100] Terminology Explanation

[0101] 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 invention pertains.

[0102] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0103] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0104] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0105] The term "antibody" (Ab) should include, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one 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 conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0106] It should be understood that the amino acid names in this article adopt the internationally accepted single-letter identifiers, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I1e (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0107] Chimeric antigen receptor (CAR)

[0108] Chimeric antigen receptors (CARs) are fusion proteins based on the intracellular domain of the TCR complex CD3ζ and the intracellular activation domain from co-stimulatory signals CD28 or 4-1BB. They overcome the disadvantage of T cell-dependent MHC antigen presentation and are known as second-generation CAR structures. The two CAR-T drugs approved in 2017 belong to this type of structure.

[0109] T-cell receptor (TCR)

[0110] The T-cell receptor (TCR) is the most complex receptor in the human body. The interaction of its six different receptor subunits determines the wide range of signals it transmits within T cells. The TCRα and TCRβ chains recognize a complex composed of a polypeptide-histocompatibility complex. The subunits that transmit TCR signals are collectively called CD3, including: a heterodimer formed by CD3ε and CD3γ, a heterodimer formed by CD3ε and CD3δ, and a homodimer of CDζ. All TCR subunits are type I transmembrane proteins, and all except CD3ζ possess immunoglobulin domains. The four different CD3 subunits in the TCR receptor possess a total of 10 immune receptor tyrosine-based activation motifs (ITAMs), which can accept a total of 20 tyrosine phosphate groups upon TCR receptor activation. In transgenic mouse experiments, it has been shown that proline-rich regions of the CD3ε intracellular segment or conformational alterations of CD3ε play a crucial regulatory role in the transmission of the complete TCR. It has been demonstrated that TCR activity can be regulated through the following mechanisms: ligand binding to TCRαβ and stabilizing the arrangement and distribution of CD3 subunits, ligand-independent TCR oligomerization, and binding to cholesterol.

[0111] TRuC structure

[0112] TCR 2 The independently developed novel T-cell therapy platform - TRuC TM The TruC structure, a chimeric antigen receptor composed of an antibody-based target antigen recognition sequence and a TCR receptor subunit, can be reprogrammed into a complete TCR complex that recognizes tumor antigens. Unlike CAR structures, TruC structures can integrate into the TCR complex to exert their effects. TruC-T cells possess the same tumor-killing activity as second-generation CAR-T cells; however, due to the absence of additional co-stimulatory signaling domains (CD28 or 4-1BB), TruC-T cells release significantly lower levels of cytokines than CAR-T cells. TruC-T cells have demonstrated antitumor activity in both hematologic malignancies and solid tumor transplantation models. Furthermore, in multiple tumor models, TruC-T cells have shown more potent antitumor activity compared to CAR-T cells.

[0113] T-cell antigen-coupled device (TAC)

[0114] Triumvira's TAC (T-cell antigen-coupled device) technology platform induces a more efficient anti-tumor response and 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-recruiting region for CD3 single-chain antibodies; and 3. a CD4 / CD8 co-receptor binding region. Preclinical experiments have shown that TAC-T technology can specifically bind to tumor cells and produce cytotoxicity, and that TAC-T cell activation is similar to that of normal T cells, avoiding the production of large amounts of cytokines. In mouse tumor transplantation models, TAC-T showed better activity than CAR-T for both solid tumors and hematologic malignancies. Furthermore, in solid tumors, TAC-T has a stronger ability to infiltrate the tumor microenvironment.

[0115] Bispecific T-cell adaptor (BiTE)

[0116] Blinatumomab, developed by Amgen, is a bispecific T-cell connector (BiTE) drug targeting CD19. It was approved by the FDA in 2014 for the clinical treatment of acute leukemia. The antibody consists of two parts: a scFv that recognizes the CD19 antigen and a scFv that recognizes the TCR (CD3e). After recognizing the tumor cell target antigen CD19, the BiTE antibody can partially induce oligomerization of endogenous TCRs on T cells using the CD3scFv, thereby activating T cells and triggering tumor killing. The approach of using BiTE to treat tumors is similar to that of TruC and TAC technologies, all inducing the activation of endogenous TCRs on T cells. Theoretically, all three have comparable activation capabilities for endogenous TCR signaling and all have significant potential value in mobilizing T cells to treat solid tumors. However, due to the systemic administration of BiTE drugs leading to poor safety profiles and their extremely short half-life in vivo, they have not yet demonstrated good efficacy in the clinical treatment of solid tumors.

[0117] Bispecific T cell activator (BiTA) structure

[0118] The terms "bispecific T-cell activator structure," "BiTA," "bispecific T-cell activator," and "-BiTA" used in this article all refer to the anti-CD3-based bispecific T-cell activator (BiTA) structure in the CAB structure, which consists of two parts: a single-domain antibody sequence (VHH) or a single-chain antibody variable region sequence (scFv) that recognizes tumor antigens, and a single-chain antibody variable region sequence that recognizes CD3.

[0119] CD3e protein

[0120] The terms "CD3e protein" and "CD3e" used in this article refer to the human CD3e protein. Its amino acid sequence is shown in SEQ ID NO.:1.

[0121] The “extracellular region of CD3e protein” mentioned in this article refers to amino acids 1-104 of the CD3e protein sequence, SEQ ID NO.:2.

[0122] The protein sequence comprises an amino acid sequence that has at least 60% homology to the amino acid sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0123] Chimeric fusion protein

[0124] The terms "chimeric fusion protein," "fusion protein," and "chimeric protein" as used herein all refer to the fusion protein expressed in T cells according to the first aspect of this invention, the structure of which is shown in Formula I below:

[0125] L-EC-H-TM-C-CD3ζ (I)

[0126] In the formula,

[0127] Each "-" independently represents a linking peptide or peptide bond;

[0128] L is an optional signal peptide sequence;

[0129] EC is a polypeptide binding domain, which can be recognized by CD3 antibodies and bind to CD3 antibodies.

[0130] The peptide-binding domain is also called the recognition-binding domain of the CD3 antibody;

[0131] H is an optional hinge area;

[0132] TM represents a transmembrane domain;

[0133] C represents a non- or co-stimulatory signaling molecule;

[0134] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0135] In another preferred embodiment, the polypeptide binding domain is derived from or derived from the CD3e protein.

[0136] In another preferred embodiment, the polypeptide binding domain is the extracellular region of CD3e or other amino acid sequences that can be recognized by CD3 antibodies.

[0137] In another preferred embodiment, the CD3 antibody is selected from the group consisting of scFV (single-chain antibody), nanobody, double-chain antibody, or variants thereof, or combinations thereof.

[0138] In another preferred embodiment, the CD3 antibody comprises the CD3 antibody segment of a BiTA molecule.

[0139] In another preferred embodiment, the polypeptide binding domain specifically recognizes and binds to the CD3 antibody.

[0140] In another preferred embodiment, the hinge region is CD8Hinge, whose amino acid sequence is SEQ ID NO.:3.

[0141] In another preferred embodiment, the transmembrane region is CD8TM, whose amino acid sequence is SEQ ID NO.:4

[0142] Chimeric CD3e and anti-CD3-based bispecific T cell activator-engineered T cells (CAB-T)

[0143] The terms "chimeric CD3e and CD3 antibody-based bispecific T cell activator edited T cells", "CAB-T cells", "CAB-T technology", "CAB structure", "CAB-T", "-CAB-T", and "-CAB" used in this article all refer to edited T cells with the following structures: The chimeric CD3e in the CAB structure consists of four components: the extracellular region of CD3e, the CD8 hinge region and the transmembrane region, the 4-1BB intracellular region, and the CD3ζ intracellular region; The anti-CD3-based bispecific T cell activator (BiTA) structure in the CAB structure consists of two parts: a single-domain antibody sequence (VHH) or a single-chain antibody variable region sequence (scFv) that recognizes tumor antigens, and a single-chain antibody variable region sequence that recognizes CD3.

[0144] The amino acid sequence of the extracellular region of CD3e is shown in SEQ ID NO.:2.

[0145] The amino acid sequence of the 4-1BB is shown in SEQ ID NO.:5.

[0146] The amino acid sequence of CD3ζ is shown in SEQ ID NO.:6.

[0147] CAIX

[0148] CAIX is a transmembrane protein expressed in various solid tumor cells. Its primary function is to maintain intracellular pH homeostasis under the hypoxic conditions common in solid tumors. CAIX expression in tumor cells is considered a marker of hypoxia in the tumor environment and a associated poor prognosis. Common tumor types expressing CAIX include cervical cancer, renal cell carcinoma, brain cancer, head and neck cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, and bladder cancer. In normal tissues, CAIX is mainly expressed in the epithelial cells of the bile ducts and small intestine, as well as gastric epithelial cells. However, unlike tumor cells, CAIX expressed in normal tissues is primarily located in the cytoplasm. Therefore, CAIX is an ideal therapeutic target for targeted therapies, including cell therapy.

[0149] HER2

[0150] HER2 is one of the most frequently researched targets in tumor immunotherapy, commonly expressed in tissues such as breast cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, urothelial carcinoma, ovarian cancer, and lung cancer. Although the HER2-targeting monoclonal antibody tricotto-mab has significantly improved the quality of life and survival of HER2-positive breast cancer patients, a large number of HER2-positive tumor patients remain unresponsive to tricotto-mab or develop resistance. Therefore, there is still a significant market demand for developing new HER2-targeting therapies. Currently, there are also reports of HER2-targeting CAR-T drugs. However, Steven Arosenberg reported severe toxic side effects in a clinical trial of a third-generation HER2-targeting CAR-T drug, resulting in patient death due to respiratory distress and severe pulmonary immune cell infiltration. Therefore, in the development of HER2-targeting cell therapies, safety must be emphasized in the drug design.

[0151] preparation

[0152] This invention provides a formulation containing the CAB-T cells described in the second aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAB-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7 Cells / ml

[0153] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0154] Therapeutic applications

[0155] This invention includes therapeutic applications using chimeric CD3e T cells as described in the first aspect of the invention and CAB-T cells as described in the second aspect of the invention. The transduced T cells can target tumor cell markers, and simultaneously, autocrine, paracrine, or BiTA-containing formulations can synergistically activate T cells, evoking a T cell immune response, thereby significantly improving their efficiency in killing tumor cells.

[0156] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue of a mammal, comprising the steps of administering chimeric CD3e T cells of the present invention or CAB-T cells as described in the second aspect of the present invention to the mammal.

[0157] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or may include solid tumors. Types of cancers treatable with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0158] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0159] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0160] The CAB-modified T cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0161] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAB into the cells, and / or iii) cryopreservation of the cells.

[0162] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing the CAB disclosed herein. The CAB-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be human, and the CAB-modified cells may be autologous relative to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0163] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0164] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAB-modified T cells of the present invention to a subject in need of the treatment.

[0165] The CAB-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides 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 invention are preferably formulated for intravenous administration.

[0166] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined through clinical trials.

[0167] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that: pharmaceutical compositions comprising T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0168] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0169] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo 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 invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0170] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The modified T cells of this invention (e.g., CAR-T20 cells) are administered to the patient via, for example, intravenous infusion.

[0171] The technical solution of the present invention has the following beneficial effects:

[0172] 1. This invention provides an immunotherapy regimen for inhibiting tumors, especially solid tumors—combining T cells expressing chimeric CD3e fusion protein with BiTA, wherein the chimeric CD3e fusion protein and BiTA bind to each other to activate T cells and target tumor cells.

[0173] 2. This invention also provides a CAB technology, which, by chimeric expression of CD3e and BiTA in T cells, allows CAB-T cells to target tumor tissue. The secreted BiTA in the tumor tissue can simultaneously activate CAB-T cells and activate the endogenous TCR of unedited T cells, thereby exerting the anti-tumor effects of CAB-T cells themselves and mobilizing the anti-tumor effects of unedited T cells. This ensures the effectiveness of CAB-T in clinical applications.

[0174] 3. Since the activation of chimeric CD3e depends on BiTA secreted by CAB-T, a small amount of BiTA released by CAB-T in tumor tissue can stimulate CAB-T to release more BiTA, which ensures the safety advantage of CAB-T in clinical applications.

[0175] 4. CAB-T can be better activated in solid tumor tissues, achieving the maximum anti-tumor effect at the tumor site and reaching the safety and efficacy of near-local tumor administration, showing great potential in the clinical treatment of solid tumors.

[0176] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0177] Example 1: Design of CAB and Control Structures

[0178] 1.1 Control group CD3e-BBζ, 1 st -CAIX-BiTA, 1 st -Structural design of CAIX-CAB and CAIX-TRuC

[0179] To verify the antitumor activity of CAB-T, in a series of experiments, we first designed four structures using nanobodies targeting CAIX: CD3e-BBζ (amino acid sequence SEQ ID NO.:7), CAIX-BiTA (amino acid sequence SEQ ID NO.:8), CAIX-CAB (amino acid sequence SEQ ID NO.:9), and CAIX-TRuC (amino acid sequence SEQ ID NO.:10). To distinguish them from the second group of CAIX-CAB structures described later, we named CAIX-BiTA and CAIX-CAB in this group 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ... st -CAIX-BiTA and 1 st -CAIX-CAB. Where, 1 st -CAIX-BiTA is an unlabeled BiTA, 1 st -CAIX-CAB is a CAB structure using CD3e-BBζ and unlabeled BiTA, while CAIX-TRuC utilizes TCR. 2 The company's platform technology comparative structure. The specific structural diagram of the above structure is shown below. Figure 1As shown.

[0180] 1.2 Structural design of CAIX-targeting nanobody assays tERBB2, CD3e-BBζ, CAIX-BiTA, CAIX-CAB, CAIX-ζ, CAIX-BBζ and CAIX-28ζ

[0181] In another set of experiments, we designed a second set of structures using nanobodies targeting CAIX, including: a truncated ERBB2 (tERBB2, comprising the ERBB2 extracellular fourth domain, transmembrane region, and FLAG tag) (amino acid sequence SEQ ID NO.:11), CD3e-BBζ, CAIX-BiTA, CAIX-CAB, CAIX-ζ (a first-generation CAR structure targeting CAIX) (amino acid sequence SEQ ID NO.:12), a second-generation CAR structure containing the 4-1BB co-stimulatory domain (CAIX-BBζ) (amino acid sequence SEQ ID NO.:13), and a second-generation CAR structure containing the CD28 co-stimulatory domain (CAIX-28ζ) (amino acid sequence SEQ ID NO.:14), totaling seven structures. In this set of structures, all structures carried a FLAG tag, and the secreted BiTA antibody carried a His tag. Specifically, as shown below... Figure 2 As shown.

[0182] 1.3 Structural design of HER2-targeting single-chain antibody assays: tERBB2, CD3e-BBζ, HER2-BiTA, HER2-CAB, HER2-ζ, HER2-BBζ, and HER2-28ζ

[0183] In the third set of experiments, we tested the antitumor activity of the CAB platform using a HER2-targeting single-chain antibody (scFv). The variable region sequence of this single-chain antibody is derived from the antibody drug Herceptin (trastuzumab) produced by Roche and Genentech, whose drug sequence-related patents have now expired. In this example, the third set of structures we designed includes: truncated ERBB2, CD3e-BBζ, HER2-BiTA, HER2-CAB (amino acid sequence SEQ ID NO.:15), HER2-ζ (amino acid sequence SEQ ID NO.:16), HER2-BBζ (amino acid sequence SEQ ID NO.:17), and the HER2-28ζ second-generation CAR structure (amino acid sequence SEQ ID NO.:18), totaling seven structures. In this set of structures, all structures also carry a FLAG tag, and all secreted BiTA antibodies also carry a His tag. Specifically, as shown below... Figure 3 As shown.

[0184] Example 2 Packaging of CAB and its control lentiviral vector

[0185] In this invention, we used lentivirus as a vector to prepare CAB-T cells. First, we prepared a lentiviral vector carrying the coding genes for CAB and its control structure. The specific lentiviral packaging process is as follows:

[0186] 1) Place 1×10⁶ seeds in a 10cm culture plate. 7 HEK 293T cells were added to 10 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C), the cells were thoroughly mixed, and the cells were incubated overnight at 37 degrees Celsius.

[0187] 2) On the second day, when the HEK 293T (ATCC, CRL-3216) cells reach about 90% confluence, replace them with serum-free DMEM;

[0188] 3) Prepare the plasmid complex with the following amounts: 8 μg plasmid DNA, 4 μg psPAX2, and 2 μg pMD2, dissolved in 1 mL opti-MEM (Gibco, 31985-070) and 42 μL PEI (Polysciences, 24765-2); vortex for 20 s. After standing at room temperature for 15 minutes, gently add the mixture along the edge to HEK293T medium and continue incubation at 37°C.

[0189] 4) After culturing for 4 hours, remove the culture medium, wash once with PBS (Hyclone, SH30256.01), and add fresh DMEM medium preheated with 2% FBS.

[0190] 5) Collect the supernatant 48h and 72h after transfection, centrifuge at 2000g for 5 minutes and discard the precipitate. Filter the supernatant through a 0.25μm filter (Sartorius, 16541-K) and add 5% PEG8000 (Sigma, 89510-1KG-F) and 0.15M NaCl (Sigma, S5150-1L) to a final concentration. Mix vigorously and incubate overnight at 4℃.

[0191] 6) Centrifuge the viral supernatant at 2000g, 4℃ for 20 minutes, remove the supernatant, and dissolve the viral precipitate in 50-100μL PBS and freeze at -80℃.

[0192] Example 3: Preparation of CAB and its control structure-engineered T cells

[0193] Once the lentiviral vector carrying the CAB structure is prepared, it can be used to infect immune cells to prepare CAB-T cells. The specific procedure for preparing CAB-T cells is as follows:

[0194] 1) Commercially available PBMCs (Sylva Biotech, SLB-HP050B) were cultured in X-VIVO 15 (LONZA, 04-418Q) containing 5‰ human serum albumin (GRIFOLS, 20% human serum albumin) at an initial cell density of 1×10⁻⁶ cells / year. 6 / mL;

[0195] 2) Add anti-CD3 / CD28 beads (Miltenyi biotec, 130-091-441) at a cell:bead ratio of 3:1, and add 1000 IU / mL IL-2 (Tetracycline Biotech, National Drug Approval Number S10970016) to activate T cell expansion;

[0196] 3) After activating the cells for 48 hours, add an appropriate amount of virus and 12 μg / mL protamine (Sigma, P4005) to infect T cells;

[0197] 4) 24 hours after lentiviral infection, aspirate the cell suspension and administer 1×10⁻⁶ cells / mL. 6 Supplement with completely fresh X-VIVO 15 medium at a concentration of cells / mL;

[0198] 5) Monitor cell density daily and supplement with T cell culture medium containing 1000 IU / ml of IL-2 as needed to maintain T cell density at 1×10⁻⁶. 6 At approximately 100 cells / mL, continue expansion for 5-10 days to complete the preparation of CAR-T cells.

[0199] Example 4: Detection of CAB-T cell positivity rate

[0200] After preparing CAB-T cells and their control group, the infection rate needs to be determined as a basis for subsequent activity analysis. Specifically, the method for detecting the CAB-T positivity rate using FLAG antibody is as follows:

[0201] 1) Take 3-5×10⁵ cells, add 200 μL of FACS buffer (PBS containing 1% FBS) to each flow cytometry tube, centrifuge at 300g for 5 min; add biotin-CAIX (sino biological, 10107-H02H) to CAIX-Truc samples to a final concentration of 100 nM, incubate at 4℃ for 20 min; add biotin-HER-2 (ACRO, HE2-H82E2) to Her2-Truc samples to a final concentration of 100 nM, incubate at 4℃ for 20 min; discard the supernatant, add 200 μL of Fixation / Permeabilization solution (BDbioscience, 554715), incubate at 4℃ for 20 min;

[0202] 2) Centrifuge at 300g for 5 min; discard the supernatant after centrifugation, add 200 μL of 1×Perm / Wash buffer (BDbioscience, 554715) to resuspend, centrifuge at 400g for 5 min; wash twice;

[0203] 3) Discard the supernatant after centrifugation, add 100 μL of anti-Flag antibody diluted with 1:1000 FACS buffer to each sample, mix the cells, and incubate at 4°C for 30 min;

[0204] 4) After incubation, add 1 mL of FACS buffer to each flow cytometry tube, centrifuge at 400 g for 5 min;

[0205] 5) Discard the supernatant after centrifugation, add 1 mL of FACS buffer to resuspend, and centrifuge at 400 g for 5 min;

[0206] 6) Discard the supernatant after centrifugation. Add 100 μL of SA-PE (Invitrogen, S866) diluted with 1:250 FACS buffer to each sample, mix the cells, and incubate at 4°C in the dark for 30 min. After incubation, add 1 mL of FACS buffer to each flow cytometry tube, centrifuge at 300 g for 5 min, discard the supernatant after centrifugation, and repeat the washing process twice.

[0207] 7) Place the sample in a flow cytometer for detection.

[0208] result:

[0209] In the first group of experiments, due to the CD3e-BBζ structure and 1 stThe CAIX-CAB structure contains a FLAG tag, allowing the use of FLAG antibodies to detect the positive rate of T cell editing based on this structure. Similarly, the positive rate of T cell transfection using CAIX-TRuC-edited T cells can be detected using biotin-labeled CAIX protein. However, due to 1 st The -CAIX-BiTA structure lacked a suitable tag, therefore its positive rate for T cell editing could not be detected. However, based on subsequent results, 1 st The positive rate of CAIX-BiTA-edited T cells met the requirements for experimental analysis. The detection results are as follows: Figure 4 As shown.

[0210] In the second and third groups of experiments, we designed FLAG tags in each structure. The positivity rate of the corresponding edited T cells could be detected using FLAG antibody labeling (the HER2-ζ generation structure did not have a FLAG tag, and biotinylated HER2 was used for positivity rate detection). NT represented non-transduced T cells (NT) and served as the negative control group. The detection results are as follows... Figure 5 and Figure 6 As shown, the differences in infection rates among different samples are within an acceptable range.

[0211] As can be seen from the above, the positive rates of T-cell transfection in each group obtained by using the structures disclosed in this invention and the prior art all meet the requirements of experimental analysis.

[0212] Example 5: Detection of CAB-T cytokine release levels

[0213] When CAB-T cells are co-cultured with tumor cells, CAB-T cells can recognize target antigens on the surface of tumor cells and become activated, releasing a large number of inflammatory cytokines. Based on this, this embodiment uses enzyme-linked immunosorbent assay (ELISA) to detect the level of cytokines released by activated CAB-T cells.

[0214] The ELISA testing process is as follows:

[0215] 1) 1×10⁻⁶ effector cells and 1×10⁻⁶ target cells each 5 200 μL / well. Centrifuge the 96-well cell culture plate that has been co-cultured overnight at 300 g for 5 min. Transfer 150 μL of supernatant / well to a new 96-well cell culture plate using a multichannel pipette. Cytokine detection was performed using the IFN-γ (invitrogen, 88-7316-88) / IL-2 (invitrogen, 88-7025-88) / TNF-α (invitrogen, 88-7346-88) assay kits.

[0216] 2) Coat the microplate with Human IFN-γ / IL-2 / TNF-αMab one day in advance. Dilute Human IFN-γ / IL-2 / TNF-αMab with PBS (1:250), add 100 μL of antibody to each well, seal the microplate with sealing film, and incubate overnight at 4°C.

[0217] 3) Washing the plate: Quickly pour out the liquid in the plate, add 200 μL of Wash Buffer to each well using a multi-channel pipette, and repeat the washing five times;

[0218] 4) Add 200 μL of 1×ELISA / ELISASPOT diluent to each well, cover with the sealing membrane, and incubate at room temperature for 60 minutes.

[0219] 5) Washing the plate: Quickly pour out the liquid in the plate, add 200 μL of Wash Buffer to each well using a multi-channel pipette, and repeat the washing five times;

[0220] 6) Prepare the Human IFN-γ ELISA Standard and set 8 gradients (unit pg / mL): 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125;

[0221] 7) Add the standard and sample to the ELISA plate, 100 μL / well. Dilute both the sample and the standard to the required concentration with 1×ELISA / ELISASPOT diluent, cover with the sealing film, and incubate at room temperature for 2 hours.

[0222] 8) Washing the plate: Quickly pour out the liquid in the plate, add 200 μL of Wash Buffer to each well using a multi-channel pipette, and repeat the washing process four times.

[0223] 9) Dilute the Human IFN-γ / IL-2 / TNF-α detection antibody with PBS (1:250), add 100 μL of antibody to each well, seal the ELISA plate with sealing film, and incubate at room temperature for 1 h;

[0224] 10) Prepare HRP-conjugated Streptavidin, dilute with PBS (1:250), and add 100 μL to each well of the microplate. Cover with sealing film and incubate at room temperature for 30 minutes;

[0225] 11) Washing the plate: Quickly pour out the liquid in the plate, add 200 μL of Wash Buffer to each well using a multi-channel pipette, and repeat the washing five times;

[0226] 12) Allow TMB Substrate to reach room temperature 30 minutes in advance, then add 100 μL to each well of the microplate. After reacting at room temperature for 5-10 minutes, add 50 μL / well stop solution.

[0227] 13) The absorbance is read by the microplate reader at the detection wavelength OD = 450 nm;

[0228] 14) Calculate the standard curve based on the concentration and OD value of the standard, and then calculate the concentration of the sample to be tested based on the standard curve. Use GraphPad Prism software to create the graph.

[0229] result:

[0230] In the first group of experiments, the release levels of inflammatory cytokines IL-2 and IFN-γ in the supernatant were measured after co-culturing CAIX-CAB-T cells or their control group cells with CAIX+HEK 293T cells or CAIX-HEK293T cells, respectively. The results are as follows: Figure 7 As shown, when CAIX-CAB-T cells and their controls were co-cultured with CAIX-HEK293T cells, no significant cytokine release was observed in any of the immune cells. However, when CAIX-CAB-T cells and their controls were co-cultured with CAIX+HEK 293T cells, T cells edited with 1st-CAIX-BiTA, 1st-CAIX-CAB, and CAIX-TRuc all exhibited time-dependent cytokine release levels, with the accumulated cytokine levels after 48 hours of co-culture significantly higher than those after 24 hours. Furthermore, when unedited T cells and CD3e-BBζ-edited T cells were co-cultured with CAIX+HEK 293T cells, no significant release of IL-2 and IFN-γ cytokines was detected. Meanwhile, it was unexpectedly found that after 48 hours of co-culture, the cytokine levels released by 1st-CAIX-CAB-T cells were significantly higher than those released by 1st-CAIX-BiTA-T cells. When CD3e-BBζ and 1st-CAIX-BiTA-edited T cells were mixed in a 1:1 ratio and then co-cultured with CAIX+HEK 293T cells, the cytokine levels released were comparable to those of 1st-CAIX-CAB-T cells. These results demonstrate the dependence of CAIX-CAB-T cell activation on the CAIX antigen and the synergistic effect of BiTA and CD3e-BBζ in promoting T cell activation; they also prove that CAB-T cells have comparable in vitro activation capacity to the control group TruC-T cells.

[0231] In the second group of experiments, the expression levels of CAIX-CAB-T cells and its control group were compared with those of CAIX+MB-231 or CAIX-MB-231 cells (CAIX expression levels were as follows). Figure 8 (As shown in A) After co-culturing, the release levels of inflammatory cytokines IL-2, IFN-γ, and TNF-α in the supernatant were detected. The results are as follows. Figure 8 As shown, when CAIX-CAB-T cells and their controls were co-cultured with CAIX-MB-231 cells, no significant cytokine release was observed in any of the cells. However, when CAIX-CAB-T cells and their controls were co-cultured with CAIX+MB-231 cells for 48 hours, CAIX-BiTA, CAIX-CAB, CAIX-BBζ, CAIX-28ζ, and CAIX-ζ edited T cells all exhibited varying degrees of activation. The data revealed that CAIX-CAB-T cells, CAIX-BiTA cells, and first- and second-generation CAR-modified T cells showed comparable IFN-γ and TNF-α release capabilities; however, in terms of IL-2 release, CAIX-CAB-T cells were significantly weaker than second-generation CAR cells, but slightly higher than CAIX-BiTA and first-generation CAR-modified T cells. The results of the second set of experiments showed that the activation of CAIX-CAB-T cells was dependent on the CAIX antigen, and that there were differences in cytokine release compared with the second-generation CAR. Specifically, the IFN-γ and TNF-α released by CAIX-BiTA-T and CAIX-CAB-T were basically equivalent to those of the second-generation CAR, while the release of IL-2 was significantly weaker than that of the second-generation CAR.

[0232] Example 6: Results of CAB-T cell activation level detection

[0233] When CAB-T cells are co-cultured with tumor cells, CAB-T cells can recognize target antigens on the surface of tumor cells and become activated. The expression levels of T cell activation marker proteins, including CD137, CD25, and CD27, are significantly upregulated. Cell proliferation capacity, represented by Ki67 expression level, increases, and the killing ability of T cells, represented by CD107a, is also enhanced. Based on this, this embodiment uses the above-mentioned staining method and flow cytometry to detect changes in the expression levels of the above-mentioned membrane surface proteins in activated CAB-T cells.

[0234] The specific cell staining procedure is as follows:

[0235] 1) 1×10⁻⁶ effector cells and 1×10⁻⁶ target cells each 5 200 μL / well. Centrifuge the 96-well cell culture plate that has been co-cultured overnight at 300 g for 5 min, add 200 μL FACS buffer to each well, and centrifuge at 300 g for 5 min.

[0236] 2) Discard the supernatant after centrifugation, add 200 μL of FACS buffer to resuspend the cells, and centrifuge at 300g for 5 min;

[0237] 3) Dilute the antibody with FACS buffer (100 μL / well)

[0238] BV421 Mouse Anti-Human CD3 (BD ​​Bioscience, 562426) 1:500 dilution

[0239] PE Mouse Anti-Human CD137 (BD Bioscience, 555956) 1:200 dilution

[0240] APC Mouse Anti-Human CD27 (BD Bioscience, 561786) 1:200 dilution

[0241] PE-cy7Mouse Anti-Human CD25 (BD Bioscience, 557741) 1:200 dilution

[0242] 4) Discard the supernatant after centrifugation, add 100 μL of antibody mix to each well, and incubate at 4°C in the dark for 30 min;

[0243] 5) Add 200 μL of FACS buffer to each well, centrifuge at 300 g for 5 min, and discard the supernatant;

[0244] 6) Discard the supernatant after centrifugation and repeat step 2.5;

[0245] 7) Discard the supernatant, add 200 μL of Fixation / Permeabilization solution (BD bioscience, 554715), and incubate at 4°C for 20 min.

[0246] 8) Centrifuge at 300g for 5 min; discard the supernatant after centrifugation, add 200 μL of 1×Perm / Wash buffer (BDbioscience, 554715) to resuspend, centrifuge at 400g for 5 min;

[0247] 9) Wash twice, and dilute the antibody FITC Mouse Anti-Flag (Biolegend, 637318) 1:1000 with FACS buffer;

[0248] 10) Centrifuge at 400g for 5 minutes; discard the supernatant after centrifugation and wash twice;

[0249] 11) Flow cytometry analysis: gate the cells using FSC and SSC to obtain the desired lymphocyte population (PBMCs). Select CD3BV421+ and Flag FITC+ cells from these cells to obtain live CAR-T cells. Then, gate the cells using untransfected PBMCs as the standard to obtain the percentage of CD137PE+ CAR-T cells.

[0250] result:

[0251] In the first group of experiments, the expression levels of CD137 and CD107a on the T cell membrane surface were detected after co-culturing CAIX-CAB-T cells or their control group cells with CAIX+HEK 293T cells or CAIX-HEK293T cells, respectively. The results are as follows: Figure 9 As shown, when CAIX-CAB-T cells and their controls were co-cultured with CAIX-HEK293T cells, the expression levels of CD137 and CD107a in CAB-T and control immune cells did not change significantly. However, after co-culturing CAIX-CAB-T cells and their controls with CAIX+HEK 293T cells for 24 hours, the expression levels of CD137 and CD107a in T cells edited by 1st-CAIX-BiTA, 1st-CAIX-CAB, and CAIX-TRuc were significantly upregulated, with 1st-CAIX-CAB-T showing a stronger upregulation than 1st-CAIX-BiTA. When unedited T cells and CD3e-BBζ-edited T cells were co-cultured with CAIX+HEK 293T cells, no significant upregulation of CD137 and CD107a was detected. When CD3e-BBζ and 1st-CAIX-BiTA edited T cells were mixed in a 1:1 ratio and co-cultured with CAIX+HEK293T cells for 24 h, the expression levels of CD137 and CD107a were significantly higher than those in the groups co-cultured with CD3e-BBζ-T and 1st-CAIX-BiTA-T alone with CAIX+HEK 293T. These results demonstrate the dependence of CAIX-CAB-T cell activation on CAIX antigen and the synergistic effect of BiTA and CD3e-BBζ in promoting T cell activation; they also prove that CAB-T cells have comparable in vitro activation capacity to the control group TruC-T cells.

[0252] In the second group of experiments, after CAIX-CAB-T cells and their control group cells were co-cultured with CAIX+HEK293T cells or CAIX-HEK293T cells for 48 h, the expression levels of CD137, CD25, CD27, and Ki67 on the T cell membrane surface were detected. The results are as follows: Figure 10As shown, when CAIX-CAB-T cells and their controls were co-cultured with CAIX-HEK 293T cells, the expression levels of CD137, CD25, CD27, and Ki67 in CAIX-CAB-T and control immune cells did not change significantly. However, when CAIX-CAB-T cells and their controls were co-cultured with CAIX+HEK 293T cells, the expression levels of CD137, CD25, CD27, and Ki67 in CAIX-BiTA, CAIX-CAB, and first-generation and second-generation CAR-edited T cells were significantly upregulated. When tERBB2 and CD3e-BBζ-edited T cells were co-cultured with CAIX+HEK 293T cells, no significant upregulation of CD137, CD25, CD27, and Ki67 was detected. These results demonstrate that CAIX-CAB-T cell activation is dependent on the CAIX antigen, and also prove that CAB-T cells have comparable in vitro activation capacity to the control group BiTA-T, first-generation CAR, and second-generation CAR cells.

[0253] In the third set of experiments, we examined the in vitro activation capacity of the HER2-CAB structure constructed using a Trastuzumab-derived scFv sequence. The results are as follows: Figure 11 As shown, the expression levels of CD137, CD25, CD27, and Ki67 on the T cell membrane surface were detected after HER2-CAB-T cells and their control group cells were co-cultured with HER2-positive SKBR3 cell line or HER2-negative RAJI cell line for 48 h. The results are as follows. Figure 12 As shown, when HER2-CAB-T cells and their controls were co-cultured with RAJI cells, the expression levels of CD137, CD25, CD27, and Ki67 in HER2-CAB-T and control immune cells did not change significantly. However, when HER2-CAB-T cells and their controls were co-cultured with HER2-positive SKBR3 cells, the expression levels of CD137, CD25, CD27, and Ki67 in HER2-BiTA, HER2-CAB, and first-generation and second-generation CAR-edited T cells were significantly upregulated. When tERBB2 and CD3e-BBζ-edited T cells were co-cultured with SKBR3 cells, no significant upregulation of CD137, CD25, CD27, and Ki67 was detected. These results demonstrate that HER2-CAB-T cell activation is dependent on the HER2 antigen, and also prove that CAB-T cells have comparable in vitro activation capacity to control BiTA-T, first-generation CAR, and second-generation CAR cells.

[0254] Example 7: Analysis of activation of unedited T cells via paracrine CAB-T.

[0255] The CAB structure was designed to enable CAB-T cells to not only activate their own anti-tumor activity upon encountering tumor cells, but also to activate unedited T cells surrounding the CAB-T cells through paracrine activation. We did not investigate the paracrine activation function of CAB-T cells, but we validated it using a transwell assay. Specifically, a 0.4 μm Transwell system was used to physically separate CAB-T cells and unedited T cells. CAB-T cells were placed in the upper chamber, while unedited T cells and tumor cells were placed in the lower chamber. The soluble BiTA secreted by CAB-T cells could freely pass through the 0.4 μm grid into the lower chamber, activating the ability of the unedited T cells in the lower layer to recognize and activate tumor cells.

[0256] The specific experimental procedure is as follows:

[0257] 1) Add 1×10 of BiTA-T and CAB-T respectively 6 Cells were resuspended in 200 μL of X-VIVO 15 medium and then added to the upper chamber of a 0.4 μm Transwell (Coning, 3413).

[0258] 2) 1 × 10⁻¹⁰ unedited T cells and 1 × 10⁻¹⁰ target cells 6 Resuspended in 500 μL of X-VIVO-15 medium and added to the lower chamber of Transwell;

[0259] 3) Carefully place the upper chamber into the lower chamber and incubate in an incubator for 48 hours;

[0260] 4) Remove the culture plate that has been co-cultured overnight and take 500 μL of cell supernatant from the lower chamber. Centrifuge at 300g for 5 min, and use a multichannel pipette to transfer 150 μL of supernatant / well to a new 96-well cell culture plate for ELISA detection of IFN-γ / IL-2 / TNF-α.

[0261] result:

[0262] In the second group of experiments ( Figure 12 Both CAIX-BiTA-T and CAIX-CAB-T secreted BiTA, which could activate the ability of unedited T cells in the lower layer to recognize CAIX-positive MB-231 tumor cells through the Transwell pores, manifested as high-level release of IFN-γ and TNF-α. Meanwhile, we found no significant change in IL-2 release levels, consistent with the results in Example 9.

[0263] From the third group of experiments ( Figure 13The results showed that HER2-CAB-T also demonstrated the same ability to paracrine activate unedited T cells to recognize tumor antigens. HER2-CAB-T cells, compared to the control tERBB2-T cells, could activate unedited T cells in the lower Transwell layer to recognize HER2-positive SKBR3 tumor cells. However, HER2-CAB-T did not help unedited T cells recognize HER2-negative RAJI cells.

[0264] Both the second and third sets of experiments showed that the CAB-T structure can activate the ability of unedited T cells to recognize tumor cells through paracrine BiTA.

[0265] Example 8: Analysis of CAB-T cell immune checkpoint expression levels and cell differentiation phenotypes

[0266] The expression levels of immune checkpoint proteins on immune cells and the differentiation phenotype of immune cells are closely related to the clinical efficacy of adoptive T cell therapy. Lower immune checkpoint expression levels and a higher proportion of memory T cells both predict better clinical response rates. We used flow cytometry to detect the effects of CAB structure on the immune checkpoint expression levels and cell phenotype of its edited T cells.

[0267] The specific analysis process is as follows:

[0268] 1) Centrifuge the 96-well cell culture plate that has been co-cultured overnight at 300g for 5 min, add 200 μL of FACS buffer to each well, and centrifuge at 300g for 5 min.

[0269] 2) Discard the supernatant after centrifugation, add 200 μL of FACS buffer to resuspend the cells, and centrifuge at 300g for 5 min;

[0270] 3) Dilute the antibody with FACS buffer to prepare an antibody mix (100 μL / well).

[0271]

[0272] 4) Discard the supernatant after centrifugation, add 100 μL of antibody mix to each well, and incubate at 4°C in the dark for 30 min;

[0273] 5) Add 200 μL of FACS buffer to each well, centrifuge at 300 g for 5 min, and discard the supernatant;

[0274] 6) Discard the supernatant after centrifugation and repeat step 2.5;

[0275] 7) Add 200 μL of Fixation / Permeabilization solution (BD bioscience, 554715) and incubate at 4°C for 20 min;

[0276] 8) Centrifuge at 300g for 5 min; discard the supernatant after centrifugation, add 200 μL of 1×Perm / Wash buffer (BDbioscience, 554715) to resuspend, centrifuge at 400g for 5 min; wash twice;

[0277] 9) Dilute the FITC Mouse Anti-Flag (Biolegend, 637318) antibody 1:1000 with FACS buffer;

[0278] 10) Centrifuge at 400g for 5 minutes; discard the supernatant after centrifugation and wash twice;

[0279] 11) Flow cytometry analysis: Gating with FSC and SSC to obtain the desired lymphocyte population. Selecting CD3BV421+ and Flag FITC+ cells yields viable CAR-T cells.

[0280] result:

[0281] The results of the second set of experiments show that ( Figure 14 After co-culturing with CAIX-positive HEK 293T cells, the expression levels of immune checkpoint proteins on the surface of CAIX-CAB-T cells, including LAG-3, PD-1, and TIM-3, were all lower than those of CAIX-28ζ second-generation CAR-T cells. The expression levels of PD-1 and TIM-3 on CAB-T cells were roughly equivalent to those of CAIX-BBζ-edited second-generation CAR-T cells, while the expression level of LAG-3 in CAB-T cells was slightly lower than that in CAIX-BBζ-edited second-generation CAR-T cells. These lower immune checkpoint expression levels demonstrate the clinical application advantages of CAB-T cells compared to second-generation CAR-T cells.

[0282] In addition, in the second set of experiments, we also used CD45RA and CCR7 to perform immune cell differentiation phenotype analysis. The differentiation marker proteins were: naive T cells (CD45RA) + CCR7 + ), central memory T cells (CD45RA) - CCR7 + ), effector memory T cells (CD45RA) - CCR7 - ), and terminal differentiation effector T cells (CD45RA) + CCR7- ).Depend on Figure 14 As shown in Figure .D, compared to second-generation CAR-T, CAIX-CAB-T cells exhibited a significantly higher proportion of newly differentiated T cells and central memory T cells, while second-generation CAR-T cells had a significantly higher proportion of effector memory T cells. A higher proportion of central memory T cells predicts better clinical efficacy, demonstrating the advantage of CAB-T over second-generation CAR-T in the differentiation stage.

[0283] From the third group of experiments ( Figure 15 The results showed that the immune checkpoint expression status and cell phenotype differentiation of HER2-CAB-T were basically consistent with the analysis results of CAIX-CAB-T. Specifically, the immune checkpoint expression level of HER2-CAB-T was lower than that of CAIX-28ζ second-generation CAR-T, and roughly equivalent to or slightly lower than that of CAIX-BBζ-edited second-generation CAR-T. Compared with second-generation CAR-T, HER2-CAB-T also exhibited a higher proportion of central memory T cell phenotypes.

[0284] Example 9: In vitro cytotoxic activity of CAB-T cells

[0285] Whether CAB-T cells possess in vitro cytotoxic activity is a key indicator for assessing the potential clinical efficacy of CAB-T therapy. To verify the tumor-killing activity of CAB-T cells, we used the LDH method for detection.

[0286] The specific experimental procedure is as follows:

[0287] 1) Set up separate experimental wells, effector cell control wells, target cell control wells, target cell maximum release wells, culture medium control wells, and volume control wells; follow the experimental procedures according to CytoTox. Non-Radioactive Cytotoxicity Assaykit (Promega, G1781) standard procedure;

[0288] 2) Set different effector-to-target ratios, i.e., effector cell number: target cell number = 0:1, 1:1, 5:1, 10:1, 20:1

[0289] 3) Cell count: 1×10⁴ target cells, 50 μL / well;

[0290] 4) For the experimental wells, add 100 μL of cells (50 μL effector cells + 50 μL target cells) to the cell culture plate at different dilution ratios of effector cells:target cells = 0:1, 1:1, 5:1, 10:1, 20:1, and set up 3 replicates.

[0291] 5) Effector cell control wells, i.e., effector cells: target cells = 0:0, 1:0, 5:0, 10:0, 20:0, with 2 replicates;

[0292] 6) Target cell control wells: Add 1×10⁴ target cells / well, 50 μL, +50 μL culture medium;

[0293] 7) Target cell maximum release well, i.e., add 1×104 target cells, 50μL, +50μL culture medium, and add 10μL lysis buffer 1h before sample collection;

[0294] 8) Control wells: Add 100 μL of culture medium.

[0295] 9) Volume control wells: Add 100 μL of culture medium. 1 hour before sample collection, add 10 μL of lysis buffer to the target cell maximum release well and incubate at 37°C.

[0296] 10) According to the designed layout, add the sample, place it on the plate and incubate at 37℃ with 5% CO2 for 24h, 36h, or 48h;

[0297] 11) Remove the assay buffer from the -20°C freezer and place it in a 4°C freezer to dissolve, taking care to avoid light. When using, add 12 ml of assay buffer to one bottle of substrate mix and mix well.

[0298] 12) Centrifuge the culture plate at 250g for 4 min, and transfer 50 μL / well of cell supernatant to a new ELISA plate;

[0299] 13) Add 50 μL of substrate mixture per well to a new microplate (protect from light; add 12 mL of assay buffer to a bottle of substrate mix and mix well);

[0300] 14) Incubate at room temperature in the dark for 30 min, then add 50 μL of stop solution per well;

[0301] 15) The absorbance is read by the microplate reader at the detection wavelength OD=490nm, and the reading is completed within 1 hour.

[0302] 16) Calculate the cell killing percentage (percentage%) based on the OD value.

[0303] Experimental well = Effectiveness-to-target ratio - Culture medium control (mean)

[0304] Spontaneous target cell count = Target cell control - Culture medium control (mean)

[0305] Spontaneous effector cell activity = Effector cell control - Culture medium control (mean)

[0306] Maximum release from target cells = Maximum release from target cells (mean) - Volume of control cells (mean)

[0307] Cell killing percentage (%) = (Experimental - Spontaneous target cell release - Spontaneous effector cell release) / (Maximum target cell release - Spontaneous target cell release)

[0308] result:

[0309] BiTA secreted by CAB-T cells can activate both the CAB-T cells themselves and surrounding unedited T cells in a target antigen-dependent manner, and kill target antigen-positive tumor cells. Simultaneously, because CAB-T cells express CD3e-BBζ, CAB-T activation not only depends on endogenous TCR activation, but CD3e-BBζ can also enhance the activation level of CAB-T cells, thereby promoting the release of more BiTA, creating a synergistic effect. Therefore, theoretically, CAB-T cells should have a stronger killing effect on tumor cells than BiTA-T cells.

[0310] In the first group of experiments, to detect the cytotoxic effect of CAIX-CAB-T cells on CAIX+HEK 293T target cells, we selected CAIX-CAB-T cells as the effector cells in the experimental group for cytotoxicity testing. Effector cells were co-cultured with target cells at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1 for 24 and 48 hours, respectively. The supernatant was then used to determine the cytotoxic ability of T cells against target cells at different effector-to-target ratios. Figure 16The results show that neither 1st-CAIX-CAB-T cells nor their control T cells had a killing effect on CAIX-HEK293T cells. However, 1st-BiTA-T cells, 1st-CAIX-CAB-T cells, CAIX-TRuC-T cells, and a mixture of CD3e-BBζ-T and 1st-BiTA-T cells all exhibited varying degrees of killing ability against CAIX+HEK 293T cells, with this killing ability increasing with the effector-to-target ratio. Furthermore, the killing effect of CAIX-CAB-T cells and their control T cells on target cells became more significant with prolonged co-culture time; the killing effect after 48 hours of co-culture was significantly stronger than that after 24 hours. CD3e-BBζ-T cells and the unedited T cell control group showed no killing effect on CAIX+HEK 293T cells. Furthermore, the killing ability of 1st-CAIX-CAB-T, CAIX-TRuC-T, and mixed T cells of CD3e-BBζ-T and 1st-BiTA-T against CAIX+HEK 293T cells was comparable to, and superior to, that of 1st-BiTA-T. Therefore, we can determine that the tumor cell killing ability of CAIX-CAB-T depends on the expression of its target antigen, and that its killing ability is comparable to that of the control group CAIX-TRuC-T cells. In addition, BiTA-T and CD3e-BBζ-T also showed a synergistic effect in killing target cells.

[0311] The second group of experiments used the same methods as the first group. We examined the killing ability of CAIX-CAB-T cells and their control cells against CAIX+MB-231 or CAIX-MB-231 tumor cells. Effector cells were co-cultured with target cells for 36 hours at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1. The supernatant was then used to determine the killing ability of T cells against target cells at different effector-to-target ratios. Figure 17 The results show that CAIX-CAB-T and its control cells did not kill CAIX-MB-231 control tumor cells; however, CAIX-CAB-T and first- and second-generation CAR-T cells targeting CAIX showed comparable killing ability against CAIX+MB-231 cells. Meanwhile, tERBB2-T and CD3e-BBζ-T control cells did not kill CAIX+MB-231 cells. This demonstrates that CAIX-CAB-T has comparable killing ability against tumor cells to first- and second-generation CAR-T cells, and this killing ability is target antigen dependent. It should be noted that due to factors such as high transfection rates or different donor cell sources, CAIX-CAB-T and CAIX-BiTA-T did not show a difference in target cell killing ability in this group of experiments.

[0312] The third group of experiments was the same as the first and second groups. We examined the killing ability of HER2-CAB-T cells and their control cells against HER2-positive tumor cells (SKBR3) or HER2-negative tumor cells (RAJI). Effector cells and target cells were co-cultured for 36 hours at effector-to-target ratios of 0:1, 1:1, 5:1, 10:1, and 20:1. The supernatant was then used to determine the killing ability of T cells against target cells at different effector-to-target ratios. Figure 18 The results show that HER2-CAB-T and its control cells did not kill HER2-negative RAJI cells; however, HER2-CAB-T and HER2-targeting first- and second-generation CAR-T cells exhibited comparable killing ability against SKBR3. Meanwhile, tERBB2-T and CD3e-BBζ-T control cells did not kill SKBR3 cells. This demonstrates that HER2-CAB-T has comparable killing power against tumor cells to first- and second-generation CAR-T, and this killing ability is target antigen-dependent. It should also be noted that due to factors such as high transfection rates or different donor cell sources (the third and second group experiments used the same T cell donor source), HER2-CAB-T and HER2-BiTA-T did not show a difference in target cell killing ability in this group of experiments.

[0313] discuss:

[0314] CAB-T technology utilizes its own secreted BiTA to simultaneously recognize chimeric CD3e or tumor antigens and endogenous CD3 on T cells, inducing tumor antigen-dependent activation of endogenous TCR and chimeric CD3e activation. The activation of both endogenous TCR and chimeric CD3e depends on the expression and secretion level of BiTA secreted by CAB-T cells. Therefore, BiTA can induce the activation of unedited T cells in the tumor microenvironment through autocrine and paracrine mechanisms. Simultaneously, after activation in tumor tissue, CAB-T cells can release higher levels of BiTA, thereby mobilizing the activation and anti-tumor effects of more unedited T cells in the tumor tissue.

[0315] Therefore, compared to TruC-T and TAC-T, CAB-T not only has the advantage of activating endogenous TCR signaling but can also mobilize the anti-tumor activity of infiltrating T cells in tumor tissue, theoretically possessing better potential for solid tumor treatment. Furthermore, unlike BiTE drugs, BiTA drugs continuously secreted by CAB-T cells solve the clinical application problem of the short half-life of BiTE monotherapy. In addition, because BiTA targeting the target antigen exerts its maximum effect at the tumor microenvironment sites reached by CAB-T cells and does not accumulate at high concentrations in non-tumor tissue sites, it has superior safety and greater clinical application potential compared to systemic administration of BiTE monotherapy.

[0316] The mechanism of action and clinical application potential of CAB-T are explained below:

[0317] 1) In tumor tissues, BiTA, which is expressed at low levels in CAB-T cells, can bind to its own endogenous TCR or CD3e-BBζ through autocrine secretion. Figure 19 B), thereby stimulating CAB-T to release more BiTA, which then fully binds to CAB-T's endogenous TCR and CD3e-BBζ. Figure 19 C) This causes CAB-T to achieve the maximum activation level at the tumor site, exerting the maximum anti-tumor effect and achieving a safety and efficacy similar to local tumor administration.

[0318] 2) By binding to and activating chimeric CD3e in CAB-T via autocrine BiTA. Figure 19 A) This endows CAB-T cells with a more sensitive proliferation and activation capacity compared to unedited T cells, further enhancing the anti-tumor activity of CAB-T cells;

[0319] 3) Activation of CAB-T (autocrine and paracrine BiTA) Figure 19 A, B, C) and unedited T cells ( Figure 19 D) This solves the problem that CAR-T cells cannot activate endogenous TCR signals, giving them the potential to treat solid tumors;

[0320] 4) CAB-T can serve as a drug synthesis factory for BiTA, solving the problem of the short half-life of BiTA in vivo; and the activation of CAB-T depends on the release level of BiTA. The two are interdependent and synergistic, jointly determining the safety and efficacy of CAB-T in clinical applications.

[0321] A schematic diagram of the mechanism of action of CAB-T is shown below. Figure 19 As shown.

[0322] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Promis Biotechnology (Suzhou) Co., Ltd. <120> Design and application of a bispecific T cell activator for T cell activation <130> P2019-1414 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 185 <212> PRT <213> Homo sapiens <400> 1 Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val 1 5 10 15 Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly 20 25 30 Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu 35 40 45 Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu 50 55 60 Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly 65 70 75 80 Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val 85 90 95 Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile Val 100 105 110 Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr Tyr 115 120 125 Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala 130 135 140 Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro 145 150 155 160 Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu 165 170 175 Tyr Ser Gly Leu Asn Gln Arg Arg Ile 180 185 <210> 2 <211> 104 <212> PRT <213> Homo sapiens <400> 2 Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val 1 5 10 15 Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly 20 25 30 Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu 35 40 45 Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu 50 55 60 Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly 65 70 75 80 Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val 85 90 95 Cys Glu Asn Cys Met Glu Met Asp 100 <210> 3 <211> 45 <212> PRT <213> Homo sapiens <400> 3 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 4 <211> 24 <212> PRT <213> Homo sapiens <400> 4 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 5 <211> 42 <212> PRT <213> Homo sapiens <400> 5 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 6 <211> 112 <212> PRT <213> Homo sapiens <400> 6 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 7 <211> 357 <212> PRT <213> Homo sapiens <400> 7 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Thr Thr 115 120 125 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 130 135 140 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 145 150 155 160 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 165 170 175 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 180 185 190 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200 205 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 210 215 220 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 225 230 235 240 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 245 250 255 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 260 265 270 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 275 280 285 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 290 295 300 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 305 310 315 320 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 325 330 335 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Asp Tyr Lys 340 345 350 Asp Asp Asp Asp Lys 355 <210> 8 <211> 387 <212> PRT <213> Homo sapiens <400> 8 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Asn Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln 50 55 60 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser Ala Gly 65 70 75 80 Gly Thr Lys Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90 95 Asp Asn Ala Lys Asn Thr Ile Leu Leu Gln Met Asn Ser Leu Lys Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 145 150 155 160 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 165 170 175 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 180 185 190 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 195 200 205 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 210 215 220 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly 260 265 270 Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser 275 280 285 Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 290 295 300 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser 305 310 315 320 Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser 325 330 335 Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 340 345 350 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 355 360 365 Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 370 375 380 Glu Leu Lys 385 <210> 9 <211> 765 <212> PRT <213> Homo sapiens <400> 9 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Thr Thr 115 120 125 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 130 135 140 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 145 150 155 160 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 165 170 175 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 180 185 190 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200 205 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 210 215 220 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 225 230 235 240 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 245 250 255 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 260 265 270 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 275 280 285 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 290 295 300 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 305 310 315 320 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 325 330 335 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Asp Tyr Lys 340 345 350 Asp Asp Asp Asp Lys Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr 355 360 365 Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr 370 375 380 Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro 385 390 395 400 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 405 410 415 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ser Ala Asn Ile Phe 420 425 430 Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg 435 440 445 Glu Leu Val Ala Val Ile Thr Ser Ala Gly Gly Thr Lys Tyr Ser Asp 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 465 470 475 480 Ile Leu Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr Trp Gly Gln Gly Thr Gln 500 505 510 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln 515 520 525 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys 530 535 540 Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys 545 550 555 560 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 565 570 575 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu 580 585 590 Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu 595 600 605 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp 610 615 620 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 625 630 635 640 Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 645 650 655 Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 660 665 670 Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val 675 680 685 Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg 690 695 700 Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe 705 710 715 720 Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met 725 730 735 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn 740 745 750 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 755 760 765 <210> 10 <211> 339 <212> PRT <213> Homo sapiens <400> 10 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Asn Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln 50 55 60 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser Ala Gly 65 70 75 80 Gly Thr Lys Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90 95 Asp Asn Ala Lys Asn Thr Ile Leu Leu Gln Met Asn Ser Leu Lys Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly Asn Glu Glu Met 145 150 155 160 Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr 165 170 175 Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln 180 185 190 His Asn Asp Lys Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly 195 200 205 Ser Asp Glu Asp His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln 210 215 220 Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala 225 230 235 240 Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu 245 250 255 Met Asp Val Met Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile 260 265 270 Thr Gly Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys 275 280 285 Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln 290 295 300 Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr 305 310 315 320 Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln 325 330 335 Arg Arg Ile <210> 11 <211> 212 <212> PRT <213> Homo sapiens <400> 11 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Ala Cys His Gln Leu Cys Ala Arg Gly His 20 25 30[[ID=3,2]] Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe Leu 35 40 45 Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro 50 55 60 Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys 65 70 75 80 Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln 85 90 95 Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 100 105 110 Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys 115 120 125 Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys Thr 130 135 140 His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln Arg 145 150 155 160 Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala Val Val Gly Ile Leu Leu 165 170 175 Val Val Val Leu Gly Val Val Phe Gly Ile Leu Ile Lys Arg Arg Gln 180 185 190 Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg Leu Leu Asp Tyr Lys Asp 195 200 205 Asp Asp Asp Lys 210 <210> 12 <211> 328 <212> PRT <213> Homo sapiens <400> 12 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Asn Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln 50 55 60 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser Ala Gly 65 70 75 80 Gly Thr Lys Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90 95 Asp Asn Ala Lys Asn Thr Ile Leu Leu Gln Met Asn Ser Leu Lys Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala 130 135 140 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 145 150 155 160 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 165 170 175 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 180 185 190 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 195 200 205 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 210 215 220 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 225 230 235 240 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 245 250 255 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 260 265 270 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 275 280 285 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 290 295 300 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 305 310 315 320 Asp Tyr Lys Asp Asp Asp Asp Lys 325 <210> 13 <211> 369 <212> PRT <213> Homo sapiens <400> 13 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn 35 40 45 Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln Ala 50 55 60<00​​​​​​​​​​​​​​​​Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro 130 135 140 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 145 150 155 160 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 165 170 175 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 180 185 190 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys 195 200 205 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 210 215 220 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 225 230 235 240 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 245 250 255 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 260 265 270 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 275 280 285 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 290 295 300 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 305 310 315 320 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 325 330 335 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 340 345 350 Leu His Met Gln Ala Leu Pro Pro Arg Asp Tyr Lys Asp Asp Asp Asp 355 360 365[[ID=**20**]] [[ID=**21**]]Lys[[ID=**22**]] [[ID=**23**]]<210> 14[[ID=**24**]] [[ID=**25**]]<211> 366[[ID=**26**]] [[ID=**27**]]<212> PRT[[ID=**28**]] [[ID=**29**]]<213> Homo sapiens[[ID=**30**]] [[ID=**31**]]<400> 14[[ID=**32**]] [[ID=**33**]]Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu[[ID=**34**]] [[ID=**35**]]1 5 10 15[[ID=**36**]] [[ID=**37**]]His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu[[ID=**38**]] [[ID=**39**]]20 25 30[[ID=**40**]] [[ID=**41**]]Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn[[ID=**42**]] [[ID=**43**]]35 40 45[[ID=**44**]] Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln Ala 50 55 60 Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser Ala Gly Gly 65 70 75 80 Thr Lys Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asn Ala Lys Asn Thr Ile Leu Leu Gln Met Asn Ser Leu Lys Pro Glu 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ile Glu Val Met Tyr 130 135 140 Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His 145 150 155 160 Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 165 170 175 Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 180 185 190 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 195 200 205 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 210 215 220 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 225 230 235 240 Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 245 250 255 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 260 265 270 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 275 280 285 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 290 295 300 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 305 310 315 320 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 325 330 335 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 340 345 350 Gln Ala Leu Pro Pro Arg Asp Tyr Lys Asp Asp Asp Asp Lys 355 360 365 <210> 15 <211> 899 <212> PRT <213> Homo sapiens <400> 15 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Thr Thr 115 120 125 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 130 135 140 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 145 150 155 160 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 165 170 175 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 180 185 190 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200 205 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 210 215 220 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 225 230 235 240 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 245 250 255 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 260 265 270 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 275 280 285 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 290 295 300 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 305 310 315 320 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 325 330 335 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Asp Tyr Lys 340 345 350 Asp Asp Asp Asp Lys Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr 355 360 365 Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr 370 375 380 Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro 385 390 395 400 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 405 410 415 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 420 425 430 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 435 440 445 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 450 455 460 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 465 470 475 480 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 485 490 495 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly 500 505 510 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 515 520 525 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 530 535 540 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 545 550 555 560 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile 565 570 575 Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg 580 585 590 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 595 600 605 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp 610 615 620 Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 625 630 635 640 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln 645 650 655 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys 660 665 670 Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys 675 680 685 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 690 695 700 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu 705 710 715 720 Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu 725 730 735 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp 740 745 750 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 755 760 765 Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 770 775 780 Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 785 790 795 800 Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val 805 810 815 Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg 820 825 830 Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe 835 840 845 Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met 850 855 860 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn 865 870 875 880 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His 885 890 895 His His His <210> 16 <211> 449 <212> PRT <213> Homo sapiens <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45 Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His 100 105 110 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 130 135 140 Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 165 170 175 Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr 210 215 220 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Arg Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 340 345 350 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 355 360 365 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 370 375 380 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 385 390 395 400 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 405 410 415 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 420 425 430 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 435 440 445 Arg <210> 17 <211> 366 <212> PRT <213> Homo sapiens <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn 35 40 45 Ser Ala Asn Ile Phe Ser Phe Ala Ser Val Ala Trp Tyr Arg Gln Ala 50 55 60 Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser Ala Gly Gly 65 70 75 80 Thr Lys Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asn Ala Lys Asn Thr Ile Leu Leu Gln Met Asn Ser Leu Lys Pro Glu 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Asn Val Asp Tyr Leu Gln Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ile Glu Val Met Tyr 130 135 140 Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His 145 150 155 160 Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 165 170 175 Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 180 185 190 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 195 200 205 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 210 215 220 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 225 230 235 240 Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 245 250 255 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 260 265 270 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 275 280 285 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 290 295 300 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 305 310 315 320 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 325 330 335 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 340 345 350 Gln Ala Leu Pro Pro Arg Asp Tyr Lys Asp Asp Asp Asp Lys 355 360 365 <210> 18 <211> 494 <212> PRT <213> Homo sapiens <400> 18 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45 Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His 100 105 110 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 130 135 140 Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 165 170 175 Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr 210 215 220 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ile Glu Val Met Tyr 260 265 270 Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His 275 280 285 Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 290 295 300 Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 305 310 315 320 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 325 330 335 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 340 345 350 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 355 360 365 Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg Asp Tyr Lys Asp Asp Asp Asp Lys 485 490

Claims

1. A genetically engineered T cell, characterized in that, The T cells express (a) BiTA, a bispecific T cell activation element based on anti-CD3, and (b) a fusion protein; The structure of the fusion protein is shown in Formula I below: L-EC-H-TM-C-CD3ζ (I) In the formula, L represents the signal peptide sequence; EC is a polypeptide-binding domain, which can be recognized and bound to CD3 antibodies. The polypeptide-binding domain is the extracellular region of CD3e protein, and its amino acid sequence is shown in SEQ ID NO.:

2. The CD3 antibody is the CD3 antigen recognition region of the BiTA. H represents the hinge area; TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; Each "-" independently represents a linking peptide or peptide bond; The structure of the anti-CD3-based bispecific T cell activation element BiTA is shown in Formula II below: L'-T1-B1-B2-T2 (Ⅱ) In the formula, L' is the signal peptide sequence; T1 is an element without a label; B1 is the tumor antigen recognition region, and the tumor antigen recognition region B1 targets CAIX; B2 is the CD3 antigen recognition region, which is a single-chain antibody variable region sequence (scFv) targeting CD3. T2 is an element without a label; Each "-" independently represents a linking peptide or peptide bond; Furthermore, the BiTA mentioned is a secretory BiTA; The expression forms of (a) and (b) are fusion expressions of the fusion proteins of structural formula I and structural formula II with protein 2A, and the amino acid sequence of the fusion expression is shown in SEQ ID NO.:

9.

2. The T cell as described in claim 1, characterized in that, The secretory BiTA mentioned is either autocrine or paracrine BiTA.

3. The T cell as described in claim 1, characterized in that, The BiTA can bind to the T cell receptor TCR.

4. A non-naturally occurring T cell population, wherein the proportion of the T cells described in claim 1 in the T cell population is ≥10%, calculated based on the total number of T cells in the T cell population.

5. A cell preparation comprising (a) T cells as described in claim 1, and / or a population of T cells as described in claim 4, and (b) a pharmaceutically acceptable carrier.

6. Use of the T cell as described in claim 1, and / or the T cell population as described in claim 4, for the preparation of a medicament for the prevention and / or treatment of cancer or tumors; in, The cancer or tumor mentioned is a solid tumor.

7. The use as described in claim 6, characterized in that, The solid tumors are selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, urinary tract tumors, thyroid cancer, pancreatic cancer, or combinations thereof.

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