B7h3-targeting car-t cell and Anti-tumor use thereof

Abstract

Provided in the present invention is an isolated recombinant nucleic acid molecule, encoding a chimeric antigen receptor (CAR) polypeptide containing a single-chain antibody (scFv) targeting a B7-H3 polypeptide. Further provided are a recombinant vector containing the recombinant nucleic acid molecule, a recombinant T lymphocyte expressing the CAR polypeptide, a method for preparing the recombinant T lymphocyte, a drug for treating cancers that contains the recombinant nucleic acid molecule, recombinant vector, and recombinant T lymphocyte, and the use of the recombinant nucleic acid molecule, recombinant vector, and recombinant T lymphocyte in the treatment of cancers.

Description

A CAR-T cell targeting B7H3 and its anti-tumor application Technical Field

[0001] This invention relates to tumor treatment, and more particularly to chimeric antigen receptor T cells. Background Technology

[0002] Chimeric antigen receptor (CAR)-T cells are an adoptive cellular immunotherapy method that combines the specific recognition of tumor cell membrane antigens by monoclonal antibodies with the killing effect of T cells. Its characteristics include high tumor antigen recognition specificity, non-MHC restriction, and the ability to be expanded in large quantities in vitro. The chimeric antigen receptor mainly consists of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region is responsible for recognizing antigens expressed on the surface of tumor cells and is mainly composed of the variable region (scFv) of specific single-chain antibodies against tumor-associated antigens (TAAs). Common transmembrane regions are mainly composed of gene sequences derived from the CD8 and CD28 transmembrane regions, and their function is to transmit the antigen signal recognized by the scFv into the cell. The intracellular region mainly consists of co-stimulatory molecules and CD3ζ. Common co-stimulatory molecules include CD137, CD28, CD27, CD134, and ICOS, which can convert the information transmitted in the transmembrane region into biological signals, thereby activating T cells to kill tumor cells.

[0003] CAR-T cell therapy has achieved significant efficacy in the treatment of hematological malignancies, with CAR-T cells targeting CD19 and BCMA already on the market for the treatment of B-lymphoblastic leukemia and other diseases. However, for acute myeloid leukemia (AML), a safe and effective CAR-T cell therapy is currently lacking, mainly due to the complexity and diversity of AML. While existing CAR-T therapies targeting CD33 and CD123 have shown good efficacy in AML treatment studies, their expression in normal bone marrow cells may lead to toxic side effects in patients. Therefore, discovering a target suitable for AML that does not affect normal hematopoietic tissue is crucial. Research on CAR-T cells in the treatment of solid tumors such as esophageal squamous cell carcinoma (ESCC) has made some progress, but the selection of suitable targets also remains a challenge.

[0004] B7-H3 (CD276; Genbank accession number: CAE47548.1) is a type I transmembrane protein belonging to the B7 immune co-stimulatory and co-inhibitory protein family. It is an important immune checkpoint member of the B7 family, existing in two subtypes: B7-H3-2Ig and B7-H3-4Ig, with B7-H3-4Ig being the predominant form found in human tissues. B7-H3 is expressed at low levels in most normal tissues but is overexpressed in various types of tumors, including colorectal cancer, prostate cancer, pancreatic cancer, squamous cell carcinoma, non-small cell lung cancer, gastric cancer, intrahepatic cholangiocarcinoma, acute myeloid leukemia, medulloblastoma, ependymoblastoma, glioblastoma, neuroblastoma, craniopharyngioma, breast cancer, ovarian cancer, laryngeal cancer, melanoma, glioma, lung cancer, and colon cancer. It is closely related to the growth, metastasis, recurrence, and poor prognosis of malignant tumors and can also mediate immune escape. These characteristics demonstrate that B7-H3 is a highly attractive target. Currently, various tumor immunotherapy modalities targeting B7H3 are being actively researched, such as monoclonal antibodies, bispecific antibodies, CAR-T therapy, and ADC drugs. B7H3 CAR-T therapy shows promising application prospects and has the potential to improve the survival rate of tumor patients with high B7H3 expression. Summary of the Invention

[0005] Previously, the inventors immunized mice with B7H3-2Ig and screened for B7H3-targeting monoclonal antibodies using antibody phage display library technology, successfully obtaining a high-affinity monoclonal antibody targeting B7H3—the mouse antibody A172-mu—and subsequently humanizing it. This study utilizes the humanized A172 antibody (A172-hu4) to construct a second-generation CAR vector using its scFv sequence. CAR-T cells targeting B7H3 are prepared via lentiviral infection. In vitro and in vivo experiments are used to evaluate the inhibitory effect of B7H3 CAR-T cells on AML and ESCC cells, laying the experimental foundation for the clinical application of B7H3 CAR-T cells and seeking a safer and more effective CAR-T cell therapy for AML and ESCC patients.

[0006] In one embodiment, the CAR polypeptide comprises, from the N to the C terminus:

[0007] (i) Single-chain antibody (scFv) targeting B7-H3 peptide;

[0008] (ii) the transmembrane portion; and

[0009] (iii) 4-1BB cytoplasmic functional regions and CD3ζ cytoplasmic functional regions connected in any order.

[0010] The present invention also provides a recombinant vector comprising a multinucleotide sequence encoding the above-mentioned CAR polypeptide.

[0011] The present invention also provides a method for preparing recombinant T cells, comprising transforming T cells with a recombinant nucleic acid molecule encoding a CAR polypeptide as described in the present invention, or a recombinant vector containing a polynucleotide sequence encoding a CAR polypeptide as described in the present invention.

[0012] The present invention also provides a recombinant T cell expressing the CAR polypeptide described herein.

[0013] The present invention also provides a medicament for treating cancer, comprising: a recombinant nucleic acid molecule encoding a CAR polypeptide as described in the present invention, and / or a recombinant vector comprising a polynucleotide sequence encoding a CAR polypeptide as described in the present invention, and / or recombinant T cells expressing the CAR polypeptide as described in the present invention, and a pharmaceutically acceptable vector.

[0014] The present invention also provides a method for treating cancer, comprising administering recombinant T cells expressing the CAR peptide described herein to a patient in need.

[0015] The present invention also provides the application of the recombinant nucleic acid molecule encoding the CAR polypeptide described in the present invention, and / or the recombinant vector containing the polynucleotide sequence encoding the CAR polypeptide described in the present invention in the preparation of recombinant T cells.

[0016] The present invention also provides the use of the recombinant nucleic acid molecule encoding the CAR polypeptide described in the present invention, and / or the recombinant vector containing the polynucleotide sequence encoding the CAR polypeptide described in the present invention, and / or the recombinant T cells expressing the CAR polypeptide described in the present invention in the preparation of drugs for treating cancer.

[0017] The present invention also provides recombinant nucleic acid molecules encoding CAR peptides as described in the present invention, and / or recombinant vectors containing polynucleotide sequences encoding CAR peptides as described in the present invention, and / or recombinant T cells expressing the CAR peptides as described in the present invention for the treatment of cancer. Attached Figure Description

[0018] Figure 1: Purity identification of the purified antibody. SDS-PAGE electrophoresis images of A172-mu and A172-hu4. The results show that the humanized antibody A172-hu4 expression vector was successfully constructed, the antibody expression was normal, and the purity was high.

[0019] Figure 2: EC50 values ​​of A172 monoclonal antibody binding to B7H3 protein before and after humanization. The binding curves of the three antibodies to human B7H3-2 Ig and B7H3-4 Ig were determined using ELISA.

[0020] Figure 3: EC50 values ​​of A172 monoclonal antibody binding to esophageal squamous cell carcinoma cells before and after humanization. Flow cytometry was used to detect the binding curves of the three antibodies to the esophageal squamous cell carcinoma cell line KYSE150.

[0021] Figure 4: Affinity detection of antibodies before and after humanization of A172. (A): Sensor diagram for SPR detection of binding of A172-hu4 to B7-H3-4 Ig protein; (B): Sensor diagram for SPR detection of binding of A172-mu to B7-H3-4 Ig protein; (C): Sensor diagram for SPR detection of binding of MGA271 to B7-H3-4 Ig protein.

[0022] Figure 5: Detection of the cytotoxic effect of A172-hu4 antibody on AML cells in vitro. (A, B): Results of LDH assay for the in vitro killing of THP-1 and U937 cells by human PBMC-mediated ADCC. The effector cell to target cell ratio was 20:1. (*P<0.05, **P<0.01, ***P<0.001)

[0023] Figure 6: Detection of the cytotoxic effect of A172-hu4 antibody on ESCC cells in vitro. (A, B): Results of LDH assay for the in vitro killing of KYSE150 and KYSE70 cells by human PBMC-mediated ADCC. The effector cell to target cell ratio was 20:1. (*P<0.05, **P<0.01, ***P<0.001)

[0024] Figure 7: Schematic diagram of B7H3 CAR and lentiviral plasmid map. (A): Schematic diagram of B7H3 CAR structure; (B): Lentiviral expression vector plasmid map. An A172-hu4 scFv-4-1BB-CD3ζ fragment was inserted between the NheⅠ and BamHI restriction sites; an A172-mu scFv-4-1BB-CD3ζ fragment was inserted between the NheⅠ and NotⅠ restriction sites.

[0025] Figure 8: Flow cytometry detection of infection efficiency and CAR expression efficiency of HEK293 cells after packaging A172-mu, hu3, and hu4-CAR viruses. (A) Flow cytometry detection of infection rate of A172-mu, hu3, and hu4-CAR in HEK293 cells; (B) Flow cytometry detection of CAR expression rate of A172-mu, hu3, and hu4-CAR in HEK293 cells.

[0026] Figure 9: Infection efficiency and CAR expression efficiency of T cells after infection with A172-hu4-CAR and A172-mu-CAR viruses. (A): Fluorescence micrographs of T cells infected with A172-hu4-CAR and A172-mu-CAR lentiviruses 96 h later (scale bar 100 μm); (B): Infection rate of A172-hu4-CAR-T and A172-mu-CAR-T cells detected by flow cytometry; (C): CAR expression rate of A172-hu4-CAR-T and A172-mu-CAR-T cells detected by flow cytometry.

[0027] Figure 10: In vitro cytotoxic effect of A172-CAR-T cells on tumor cells. (A): LDH assay for the cytotoxic effect of A172-hu4-CAR-T and A172-mu-CAR-T cells on AML cell lines THP-1 and U937, and ESCC cell lines KYSE70 and KYSE150; (B): IFN-γ secretion in the supernatant of A172-CAR-T cells and tumor cells co-cultured; (C): IL-2 secretion in the supernatant of A172-CAR-T cells and tumor cells co-cultured. (*P<0.05, **P<0.01, ***P<0.001)

[0028] Figure 11: A172-CAR-T cells exhibit strong killing activity against AML in vivo. (A): Flowchart for constructing a mouse model of disseminated AML xenografts; (B): Imaging image of the mouse model of disseminated AML xenografts; (C): Statistical line graph of fluorescence expression levels in mice at different time points; (D): Curve of mouse body weight change during CAR-T treatment; (E): Results of complete blood count in mice; (F): Organ coefficients in mice. (*P<0.05, **P<0.01, ***P<0.001)

[0029] Figure 12: A172-CAR-T cells exhibit strong killing activity against ESCC in vivo. (A): Tumor volume growth curve in the KYSE150 esophageal squamous cell carcinoma xenograft model (PBS, NT group n=4, A172-mu-CAR-T, A172-hu4-CAR-T group n=5); (B): Actual tumor images of each group in the KYSE150 esophageal squamous cell carcinoma xenograft model (PBS, NT group n=4, A172-mu-CAR-T, A172-hu4-CAR-T group n=5); (C): Tumor weight after CAR-T cell treatment (PBS, NT group n=4, A172-mu-CAR-T, A172-hu4-CAR-T group n=5); (D): Mouse body weight change curve during CAR-T treatment (PBS, NT group n=4, A172-mu-CAR-T, A172-hu4-CAR-T group n=5). (*P<0.05, **P<0.01, ***P<0.001) Detailed Implementation

[0030] While various embodiments and aspects of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments and aspects are merely illustrative. Many variations, modifications, and substitutions can be made by those skilled in the art without departing from the spirit of the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used in the practice of the invention.

[0031] Unless otherwise specified, all technical and academic terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Additionally, some terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications cited herein are incorporated by reference as shown in their entirety.

[0032] Unless otherwise stated, nucleic acids are written from left to right in the 5′ to 3′ direction; amino acid sequences are written from left to right in the direction from amino (N) to carboxyl (C).

[0033] This invention provides an isolated recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a CAR polypeptide. The recombinant nucleic acid molecule optionally comprises a nucleotide sequence encoding a signal peptide.

[0034] The term "signal peptide" herein refers to its common meaning in the art, referring to a peptide having approximately 5-30 amino acids. Signal peptides are located at the N-terminus of newly synthesized proteins that form part of a secretory pathway. Proteins of the secretory pathway include, but are not limited to, proteins located within organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), proteins secreted from the cell, or proteins inserted into the cell membrane. In one embodiment, the signal peptide shown is, for example, as shown in SEQ ID NO:13.

[0035] As used herein, the term "isolated" when referring to nucleic acids or proteins means that the nucleic acid or protein is substantially free of its natural binding environment. "Substantially free" means that at least 50%, advantageously at least 70%, more advantageously at least 80%, and even more advantageously at least 90% of these substances are absent. Biological components that have been "isolated" include those purified by conventional purification methods. The term also includes recombinant nucleic acids or proteins, as well as nucleic acids or peptides synthesized by chemical methods.

[0036] As used herein, the term "recombinant" refers to cells, nucleic acids, proteins, or carriers that have been modified or are the result of laboratory methods. Thus, for example, recombinant proteins include proteins produced by laboratory methods. Recombinant proteins may include amino acids not found in their natural (non-recombinant) forms, or may include modified amino acid residues, such as those that have been labeled.

[0037] As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, their single-stranded or double-stranded polymers, and their complementary sequences. The term "polynucleotide" or "nucleotide sequence" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of nucleotide sequences in this article include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. As used herein, nucleic acid also refers to nucleic acids having the same basic chemical structure as naturally occurring nucleic acids. Such analogs have modified sugars and / or modified ring substituents, but retain the same basic chemical structure as naturally occurring nucleic acids.

[0038] As used herein, the terms “protein,” “peptide,” “polypeptide,” and “amino acid sequence” are used interchangeably to refer to polymers of any length, such as two or more amino acid residues. The term also includes amino acid polymers that are naturally or artificially modified; for example, through disulfide bonding, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation and modification, such as conjugation to tags or biologically active components. Conventional single-letter or three-letter amino acid residue encodings are used herein.

[0039] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids—an α-carbon, carboxyl, amino, and R group bound to hydrogen—such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid mimics are compounds that have a structure different from the typical chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids. In this document, amino acids may be represented by known three-letter symbols or by single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted single-letter symbols.

[0040] As used herein, the CAR peptide is a CAR peptide targeting the B7-H3 peptide. In one embodiment, the CAR peptide comprises: a single-chain antibody (scFv) targeting the B7-H3 peptide, a transmembrane portion, and a cytoplasmic functional region, wherein the cytoplasmic functional region comprises a 4-1BB cytoplasmic functional region and a CD3ζ cytoplasmic functional region, wherein the 4-1BB cytoplasmic functional region and the CD3ζ cytoplasmic functional region can be connected in any order, for example, from N to C, the 4-1BB cytoplasmic functional region and the CD3ζ cytoplasmic functional region, or the CD3ζ cytoplasmic functional region and the 4-1BB cytoplasmic functional region.

[0041] As used herein, the term "antibody" refers to a polypeptide or portion thereof that specifically binds to and recognizes antigens and contains a framework region derived from an immunoglobulin gene. Typically, the antigen-binding region of an antibody plays a significant role in determining the specificity and affinity of the binding. In some embodiments, antibodies or antibody fragments may be derived from various organisms, including humans, mice, rats, hamsters, camels, etc. Antibodies or fragments thereof, their preparation, and their use are well known and disclosed, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.

[0042] scFv is typically a fusion protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin are linked by a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility and contains serine or threonine for solubility. The linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa. The linker connecting VH and VL can be any suitable linker known in the art. In one embodiment, the VH and VL of the targeting molecule scFv are linked directly or via a linker in any order from N to C-terminus, for example: VH-VL, VL-VH, VH-linker-VL, or VL-linker-VH.

[0043] In one embodiment, the scFv comprises VL and VH, wherein (a) the VL comprises the light chain CDR1 (CDR-L1) shown in SEQ ID NO:1, the CDR-L2 shown in SEQ ID NO:2, and the CDR-L3 shown in SEQ ID NO:3; and (b) the VH comprises the heavy chain CDR1 (CDR-H1) shown in SEQ ID NO:4, the CDR-H2 shown in SEQ ID NO:5, and the CDR-H3 shown in SEQ ID NO:6.

[0044] In one embodiment, the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 7 or 9, and / or the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10. In one embodiment, the VL comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7 or 9, and / or the VH comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8 or 10. In a preferred embodiment, the scFv comprises or is composed of VL-linker-VH from the amino terminus to the carboxyl terminus.

[0045] Any linker known to those skilled in the art can be used in this invention. The linker moiety can be a peptide. The linker does not form an antigenic epitope. Typical amino acid residues used for linkers are glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, etc. Examples of such known linker moiety include, but are not limited to, (G). n S(G) n Where n = 4, 5, 6 or 7 (SEQ ID NO: 37-40), or (G4S) n (SEQ ID NO:41), where n = 3, 4, 5, 6, 7, 8 or 9. In a preferred embodiment, the connector is (G4S)3 (SEQ ID NO:14).

[0046] Preferably, the scFv comprises or consists of SEQ ID NO:15 or 16, and more preferably comprises or consists of SEQ ID NO:16.

[0047] In this invention, the terms "transmembrane portion" and "transmembrane region" are used interchangeably and have meanings commonly known to those skilled in the art, referring to the portion of a transmembrane protein that connects the extracellular and intracellular regions of the protein, spanning the cell membrane, typically an α-helix structure of about 20-25 amino acid residues. The amino acids constituting the transmembrane portion of a protein are predominantly hydrophobic. The transmembrane portions described herein are capable of anchoring proteins encoded by recombinant CAR genes provided herein and in embodiments thereof to biological membranes (e.g., the cell membrane of T cells). Any transmembrane domain capable of anchoring proteins encoded by recombinant CAR genes provided herein and in embodiments thereof is included in this invention.

[0048] In one embodiment, the transmembrane portion of the CAR peptide of the present invention is selected from the transmembrane portion of CD molecules, such as the transmembrane portion of CD30 molecules, the transmembrane portion of CD8 molecules, the transmembrane portion of CD28 molecules, the transmembrane portion of 41BB molecules and the transmembrane portion of CD3ζ molecules, preferably selected from the transmembrane portion of CD8 molecules and the transmembrane portion of CD28 molecules.

[0049] In one embodiment, the transmembrane portion of the CAR polypeptide of the present invention is the transmembrane portion of the CD8 molecule. The term "CD molecule transmembrane portion" as used herein includes any recombinant or naturally occurring form of the CD molecule transmembrane domain, or variants or homologues that retain the activity of the CD molecule transmembrane domain (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD molecule transmembrane domain). In some aspects, said variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the naturally occurring CD molecule transmembrane domain polypeptide in the range of the whole sequence or a partial sequence (e.g., 50, 100, 150, or 200 consecutive amino acid portions).

[0050] Sequence identity can be determined using commercially available computer programs that employ any suitable algorithm to calculate the percentage of similarity between two or more sequences, for example, using default parameters. A typical example of such a computer program is CLUSTAL. More advantageously, the BLAST algorithm is used with the parameters set to default values. A detailed description of the BLAST algorithm is available on the National Center for Biotechnology Information (NCBI) website.

[0051] In one embodiment, the CD8 transmembrane domain is a protein, its homologue, or a functional fragment comprising the amino acid sequence shown in SEQ ID NO:17. Optionally, the nucleic acid sequence encoding the transmembrane portion of the CD8 molecule comprises, for example, the sequence shown in SEQ ID NO:34.

[0052] In one embodiment, the transmembrane portion of the present invention (e.g., the CD8 transmembrane domain) is directly or via a spacer subregion connected to the C-terminus of the scFv of the B7-H3 polypeptide (e.g., the C-terminus of the heavy chain variable region or the light chain variable region).

[0053] In one embodiment, a spacer region (also referred to as a hinge region) may be present between the scFv of the B7-H3 peptide and the transmembrane portion.

[0054] In one embodiment, the transmembrane portion further includes a spacer region (also referred to as a hinge region) connecting the scFv and the transmembrane domain, such as the hinge region of a CD8 molecule. The term "spacer region" as used herein refers to a peptide connecting a target molecule to the transmembrane portion. In some embodiments, the spacer region connects a heavy chain constant region to the transmembrane portion. In some embodiments, the spacer region includes an Fc region, such as IgG Fc. Examples of spacer regions include, but are not limited to, immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) and / or immunoglobulin molecules or fragments thereof that include mutations affecting Fc receptor binding (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the spacer region is a fragment of IgG (e.g., IgG4) wherein the fragment includes a deletion of the CH2 domain. The spacer region may be a peptide linker. In some embodiments, the recombinant nucleic acid molecule of the present invention does not include the nucleotide sequence encoding the spacer region.

[0055] In this invention, the terms "cytoplasmic region" and "intracellular region" are used interchangeably, including an amino acid sequence capable of providing primary signaling for the binding of a response antigen to the antigen-binding region provided in the embodiments herein, resulting in activation and / or proliferation (cell division) of T cells expressing the CAR gene.

[0056] The intracellular region of the CAR polypeptide of the present invention includes a 4-1BB cytoplasmic functional region and a CD3ζ cytoplasmic functional region, wherein the 4-1BB cytoplasmic functional region and the CD3ζ cytoplasmic functional region can be connected in any order.

[0057] 4-1BB (also known as CD137) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is another important co-stimulatory molecule mediating T cell activation, besides CD28 / B7. It is mainly distributed on the surface of activated CD4-positive T cells, CD8-positive T cells, and NK cells. The cytoplasmic functional region of the 4-1BB molecule contains a conserved sequence of 5 amino acids, which can mediate the second signal for T cell activation. The cytoplasmic functional region of the 4-1BB molecule described in this invention refers to all or part of the cytoplasmic region of the 4-1BB molecule capable of mediating T cell activation. Preferably, the cytoplasmic functional region of the 4-1BB molecule comprises or is composed of the amino acids shown in SEQ ID NO:18; more preferably, the nucleic acid sequence encoding the cytoplasmic functional region of the 4-1BB molecule comprises or is composed of the sequence shown in SEQ ID NO:35.

[0058] The CD3ζ molecule is a T cell stimulating factor and a member of the T cell co-receptor complex. The CD3 cytoplasmic region contains three ITAMs, which can trigger T cell division and cytokine release. The cytoplasmic functional region of the CD3ζ molecule described in this invention refers to all or part of the cytoplasmic region of the CD3ζ molecule that contains the three ITAMs and can trigger T cell division and cytokine release. Preferably, the cytoplasmic functional region of the CD3ζ molecule comprises or is composed of the amino acids shown in SEQ ID NO:19; more preferably, the nucleic acid sequence encoding the cytoplasmic functional region of the CD3ζ molecule comprises or is composed of the sequence shown in SEQ ID NO:36.

[0059] In one embodiment, the present invention provides an isolated recombinant nucleic acid molecule comprising, from the 5' to 3' direction, a nucleotide sequence encoding the following: a single-chain antibody (scFv) targeting a B7-H3 polypeptide, a transmembrane portion, a 4-1BB cytoplasmic functional region, and a CD3ζ cytoplasmic functional region, wherein the 4-1BB cytoplasmic functional region and the CD3ζ cytoplasmic functional region can be connected in any order, for example, from the N to C direction, the 4-1BB cytoplasmic functional region and the CD3ζ cytoplasmic functional region, or the CD3ζ cytoplasmic functional region and the 4-1BB cytoplasmic functional region.

[0060] Optionally, the recombinant nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide. In one embodiment, the signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0061] In one embodiment, the present invention provides an isolated recombinant nucleic acid molecule, which, from the 5' to 3' orientation, comprises nucleotide sequences encoding a single-chain antibody (scFv) targeting a B7-H3 polypeptide, a transmembrane portion, a 4-1BB cytoplasmic functional region, and a CD3ζ cytoplasmic functional region. Optionally, the recombinant nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide.

[0062] In one embodiment, the recombinant nucleic acid molecule contains, from 5' to 3', a nucleotide sequence encoding the following:

[0063] (1) A scFv targeting the B7-H3 peptide, comprising a VL-linker-VH, wherein the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 7 or 9, and the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10; optionally, the linker comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14; preferably, the scFv comprises or is composed of SEQ ID NO: 15 or 16.

[0064] (2) The transmembrane portion of the CD8 molecule, preferably comprising or consisting of the amino acid sequence shown in SEQ ID NO:17.

[0065] (3) 4-1BB cytoplasmic functional region and CD3ζ cytoplasmic functional region, which contain or consist of the amino acid sequences shown in SEQ ID NO:18 and 19.

[0066] In one embodiment, the CAR polypeptide comprises the sequence shown in SEQ ID NO:21 or 22, or the sequence shown in amino acids 22-490 of SEQ ID NO:21 or 22, or the sequence shown in amino acids 22-490 of SEQ ID NO:21 or 22.

[0067] In one embodiment, the recombinant nucleic acid molecule comprises or consists of the sequence shown in SEQ ID NO:32 or 33, or the sequence shown in nucleotides 64-1473 of SEQ ID NO:32 or 33.

[0068] The present invention also provides a recombinant vector comprising the polynucleotide sequence of the recombinant nucleic acid molecule encoding the CAR polypeptide described in the present invention, which is, for example, but not limited to, expression vectors such as baculovirus expression vectors, plasmids, viral vectors such as lentivirus or retrovirus vectors, bacterial vectors, protozoan vectors, insect vectors, yeast vectors, and mammalian cell vectors.

[0069] The vector may include regulatory sequences that regulate the expression of the nucleic acid encoding the fusion protein, such as promoters, enhancers, etc. The promoter may be any promoter that is functional in mammalian cells, such as including but not limited to T7, CMV promoters, etc.

[0070] The methods for preparing and / or administering vectors, recombinants, or plasmids to express the recombinant nucleic acids of the present invention in vivo or in vitro can be any desired method. As used herein, an "expression vector" is a recombinant or synthetically produced nucleic acid construct having a series of designated nucleic acid elements that allow specific nucleic acids to be transcribed in host cells. For example, in addition to encoding the nucleic acid sequence to be expressed, the expression vector may include replication and control sequences derived from host-compatible sequences used for expression, such as enhancer sequences, stabilizing sequences, and signaling sequences that allow protein secretion, as well as selection markers that confer a selectable phenotype to the transfected cells. The CAR recombinant gene of the present invention can be incorporated into plasmids, viruses such as lentiviruses, or retroviral vectors. The vector of the present invention can be a lentiviral plasmid vector, such as pLent-EF1a, or a lentiviral vector.

[0071] According to the present invention, any vector capable of expressing the polypeptides described herein can be used. In some embodiments, the polypeptides of the present invention can be expressed in vitro (e.g., using a cell-free expression system) and / or in cultured cells grown in vitro. For such applications, any vector that allows expression of the polypeptide in vitro and / or in cultured cells can be used.

[0072] The present invention also provides a method for preparing recombinant T lymphocytes, comprising transforming T lymphocytes with a vector expressing the CAR polypeptide described in the present invention, thereby causing the transformed T lymphocytes to express the CAR polypeptide.

[0073] In one embodiment, the method includes transforming T lymphocytes with a recombinant nucleic acid molecule encoding a CAR polypeptide as described in this invention, or a recombinant vector comprising a polynucleotide sequence encoding a CAR polypeptide as described in this invention.

[0074] In one implementation, the T lymphocytes are CD3 + T lymphocytes.

[0075] The term "transformation" refers to a method of introducing nucleic acid molecules or proteins into cells. Nucleic acid is introduced into cells using non-viral or virus-based methods. Nucleic acid molecules can be gene sequences encoding complete proteins or functional portions thereof. Non-viral transformation methods include any suitable method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Examples of non-viral transformation methods include calcium phosphate transfection, liposome transfection, nuclear staining, sonication, transfection by heat shock, magnetic transfection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells by electroporation according to standard procedures known in the art. For virus-based transformation methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, and adeno-associated virus vectors. In some embodiments, nucleic acid molecules are introduced into cells using retroviral vectors according to standard procedures known in the art. Methods of transforming cells with vectors are well known in the art and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)). The term "conversion" also refers to the introduction of proteins from the external environment into cells. Typically, protein conversion relies on peptides or proteins that can cross the cell membrane attaching to the protein of interest.

[0076] In one embodiment, the transformed T lymphocytes are expanded in vitro for at least 7, 8, 9, 10, 11, 12, 13, or 14 days. Methods for culturing and expanding T lymphocytes are well known to those skilled in the art, for example, see [link to relevant documentation]. Human T-Activator CD3 / CD28 (Cat. Nos: 11131D, 11132D and 11161D, Life Technologies AS, Norway).

[0077] The present invention also provides a recombinant T lymphocyte expressing the CAR polypeptide described herein. In one embodiment, the recombinant T lymphocyte is a recombinant CD3+ T lymphocyte. + T lymphocytes.

[0078] In one embodiment, the T lymphocytes can be obtained using the method for preparing recombinant T lymphocytes according to the present invention, for example, by transforming T lymphocytes with a recombinant nucleic acid molecule encoding a CAR polypeptide as described in the present invention, or a recombinant vector containing a polynucleotide sequence encoding a CAR polypeptide as described in the present invention, and optionally by in vitro expansion, as described above.

[0079] The present invention also provides a medicament for treating cancer, comprising: a recombinant nucleic acid molecule encoding a CAR polypeptide as described in the present invention, or a recombinant vector comprising a polynucleotide sequence encoding a CAR polypeptide as described in the present invention, and / or recombinant T cells expressing the CAR polypeptide as described in the present invention, optionally comprising a pharmaceutically acceptable vector.

[0080] "Pharmaceutically acceptable carrier" refers to a substance that facilitates the delivery and absorption of an active substance by a subject, and may be included in the compositions of the present invention without causing significant toxic side effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants, etc. Those skilled in the art will understand that other drug carriers may be used in the present invention.

[0081] If desired, the drug may be contained in a box, vial, or dispenser, which may, for example, contain one or more unit doses of the recombinant nucleic acid molecule, recombinant vector, and / or recombinant T lymphocytes described in this invention. The box, vial, or dispenser may be accompanied by instructions for use.

[0082] The present invention also provides a method for treating cancer, comprising administering recombinant T cells expressing the CAR peptide described herein to a subject in need. In one embodiment, the recombinant T cells are recombinant CD3+ cells. + T cells.

[0083] As used herein, the term "treatment" refers to the relief of at least one symptom of cancer. This term includes administering medication to a subject and / or applying one or more recombinant nucleic acids, vectors, or T cells described herein and / or drugs comprising them to provide management or treatment of cancer. For the purposes of this disclosure, "treatment" may, but does not necessarily, provide a cure; rather, it means that "treatment" can be a form of disease management. As used herein, "treatment" of a subject with cancer means that the subject's cancer is partially or completely eliminated, or remains stable and no longer progresses after treatment. Treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the occurrence of potential cancer and / or preventing the progression or worsening of cancer. Preventing cancer includes mitigating or eliminating one or more risk factors that contribute to cancer; because it is generally not possible to determine whether cancer has never occurred, prevention also includes reducing the risk of developing or having cancer. When used herein to address harmful proliferating cells (including cancer), "treatment" includes the partial or complete destruction of said harmful proliferating cells, but with minimal impact on normal cells.

[0084] In one implementation, administration of the recombinant T cells induced an immune response against cancer. The term "inducing an immune response" encompasses both preventative purposes, such as providing protective immunity and / or inoculating subjects, and therapeutic purposes, such as evoking a desired immune response in subjects in need.

[0085] As used herein, "patient" or "object of need" refers to an organism that suffers from or is susceptible to a disease or condition that can be treated by administration of the compositions or pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or object is a human.

[0086] As used herein, “therapeutic effective amount” or “therapeutic effective dose” means an amount of a pharmaceutical agent, compound, or material in a preparation that is at least sufficient to produce a therapeutic effect in a subject. The exact amount depends on the therapeutic purpose and can be determined by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0087] As used herein, the term “administration” means to be given by any appropriate route, including, for example, intravenous, intraarterial, intraventricular, and intralymphatic tissue.

[0088] In some embodiments, the method includes administering the T lymphocytes to the patient multiple times. The dosage and intervals of administration allow the T cells to persist in the body and kill cancer cells, and the post-infusion cytokine storm is mild.

[0089] In some implementations, the method includes administering the T lymphocytes to a patient two, three, four or more times, wherein each administration may be spaced at appropriate intervals, such as 7-60 days, or about 10, about 15, about 20, about 25, about 30, about 35, about 40 or about 50 days.

[0090] In some implementations, the dose of T lymphocytes administered each time is 1 × 10⁻⁶. 5 Cells / kg body weight - 1×10 8 Cells / kg body weight, for example, about 5 × 10 5 Cells / kg body weight, approximately 1×10 6 Cells / kg body weight, approximately 5 × 10 6 Cells / kg body weight, approximately 1×10 7 Cells / kg body weight, approximately 5 × 10 7 Cells / kg body weight.

[0091] In some embodiments, the T lymphocytes described in this invention are autologous T lymphocytes.

[0092] In some embodiments, the T lymphocytes described in this invention are allogeneic T lymphocytes.

[0093] In one embodiment, the method for treating cancer in the target body according to the present invention includes the following steps:

[0094] (i) Obtain the subject's T lymphocytes;

[0095] (ii) Transforming the T lymphocytes of step (i) with the recombinant nucleic acid encoding the CAR polypeptide as described in this invention or the recombinant vector comprising the polynucleotide sequence encoding the CAR polypeptide as described in this invention, optionally expanding the transformed T lymphocytes for, for example, at least 7-14 days, such as at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days; and

[0096] (iii) Administer the transformed, optionally expanded T lymphocytes obtained in step (ii) to the subject, optionally multiple times, at intervals of, for example, 7-30 days, at a dose of, for example, about 1 × 10⁻⁶. 6 Cells / kg body weight.

[0097] The present invention also provides the application of the recombinant nucleic acid molecule encoding the CAR polypeptide described in the present invention, and / or the recombinant vector containing the polynucleotide sequence encoding the CAR polypeptide described in the present invention in the preparation of recombinant T lymphocytes.

[0098] The present invention also provides the use of the recombinant nucleic acid molecule encoding the CAR polypeptide described in the present invention, and / or the recombinant vector containing the polynucleotide sequence encoding the CAR polypeptide described in the present invention, and / or the recombinant T cells expressing the CAR polypeptide described in the present invention in the preparation of drugs for treating cancer.

[0099] The present invention also provides recombinant nucleic acid molecules encoding CAR peptides as described in the present invention, and / or recombinant vectors containing polynucleotide sequences encoding CAR peptides as described in the present invention, and / or recombinant T cells expressing the CAR peptides as described in the present invention for the treatment of cancer.

[0100] In this invention, the cancer is a B7-H3 positive cancer.

[0101] As used herein, B7-H3 positive cancer refers to cancer that uses B7-H3 as a biomarker or therapeutic target, including hematologic malignancies and solid tumors. For example, the B7-H3 positive cancer may be selected from esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer. In one embodiment, the cancer is esophageal cancer.

[0102] As used herein, “optional” or “optional” means that the event or situation described below occurs or does not occur, and the description includes both the occurrence and non-occurrence of the event or situation.

[0103] The present invention is further described below through specific embodiments, but the present invention is not limited to the following embodiments. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions.

[0104] Example 1: Preparation and purification of antibodies

[0105] 1. Preparation of mouse antibodies

[0106] 1.1) Mouse immunization

[0107] Mice were immunized with B7-H3 2Ig protein (purchased from Beijing Baipusai Biotechnology Co., Ltd., Cat.#B73-H52E2) with a purity of over 95% and free of endotoxin. Three 6-8 week old female Balb / c mice were selected and injected subcutaneously at multiple sites on their backs, with each mouse receiving 100 μg of protein per injection, for a total of 3 times, with each injection spaced 2 weeks apart. After the last booster immunization, blood was collected to measure the serum antibody titer. If the titer reached 80,000, a pulse immunization was administered via intraperitoneal injection. The spleen of the mice was harvested on the 3rd day.

[0108] 1.2) Phage display library construction and lead antibody screening

[0109] RNA was extracted from spleen cells of immunized mice and reverse transcribed. A mouse immune antibody library was constructed using a primer-based amplification method, and the library size was determined. The correct insertion rate of antibody genes was verified by single-clone sequencing analysis. The mouse immune library was screened using an immunosorbent assay tube and magnetic bead screening system, employing a combination of solid-phase and liquid-phase screening to enrich specific Fab antibodies. Positive clones binding to the antigen were obtained after antibody sequencing and ELISA screening, and full-length antibodies were constructed using pCDNA3.1. Finally, antibody A172 was selected and named A172-Mu (VL and VH are shown in SEQ ID NO:7 and 8, respectively).

[0110] 2. Preparation of humanized antibodies

[0111] Using the IMGT database (http: / / www.imgt.org / IMGTrepertoire / Proteins / ), the complementarity-determining regions (CDRs, SEQ ID NO:1-6) in the variable regions of the light and heavy chains of the murine antibody were preserved. The backbone region (FR) was compared with the human backbone region, and the human antibody backbone region (FR) with the highest homology was used to replace the murine antibody backbone regions (VL and VH, as shown in SEQ ID NO:9 and 10, respectively). The antibody light and heavy chain constant regions were replaced with the human light chain constant region (κ, Uniprot P01834, as shown in amino acids 109-215 of SEQ ID NO:11) and the IgG1 heavy chain constant region (Uniprot P01857, as shown in amino acids 123-452 of SEQ ID NO:12). The replaced sequences were then codon-optimized and synthesized by Shanghai Sangon Biotech Co., Ltd., and ligated into the pTT5 vector to construct a full-length eukaryotic expression vector plasmid named A172-Hu4. The antibody type was IgG1κ.

[0112] 3. Antibody Expression and Purification

[0113] The plasmid transformation and extraction methods are the same as those for the plasmid extraction kit.

[0114] (1) Prepare two centrifuge tubes and add 5 mL of HEK 293 medium to each tube. Add 200 μg of the heavy chain and light chain plasmid of the antibody to one tube. Add 600 μl of PEI to the other tube and incubate for 5 min.

[0115] (2) After thoroughly mixing the plasmid and PEI culture medium, incubate them together for 20 min. Then add the mixture to 100 mL of culture medium containing HEK293 cells and place the shake flask on a shaker in a 37°C, 5% CO2 incubator.

[0116] (3) On the second day, the temperature of the incubator was lowered to 32°C, and 10% of the system of SMS293-SUPI feed and 6 nM of glutamine were added to the shake flask on the second and fourth days, respectively.

[0117] (4) On day 5, collect the supernatant of HEK 293 cell culture, centrifuge at 4500 rpm for 10 min, and then filter the supernatant through 0.45 μm and 0.22 μm filters in sequence.

[0118] (5) Rinse the HiTrap Heparin HP affinity chromatography column with 20 mL PBS, then slowly filter the supernatant through the column, and rinse the affinity chromatography column again with 20 mL PBS. Then elute the antibody from the column with 1 mL 0.1 M glycine (pH 2.7), and immediately add an appropriate volume of 1 M Tris-HCl (pH 8.0) to the EP tube to adjust the pH of the antibody solution to 7.0.

[0119] (6) Transfer the purified antibody solution into a dialysis bag, place the dialysis bag in PBS, rotate overnight, and change the PBS every 4 hours. After changing the PBS 3 times, the antibody solution in the dialysis bag can be collected. Use Nanodrop to detect the plasmid concentration and check whether the absorbance ratios of A260 / A280 and A260 / A230 are within the normal range.

[0120] (7) The position and expression level of the antibody bands were observed by SDS-PAGE electrophoresis, and the purity of the bands was calculated using ImageJ. The results are shown in Figure 1, which shows that the humanized antibody A172-hu4 expression vector was successfully constructed, the antibody expression was normal, and the purity was high.

[0121] Example 2: Antibody Detection

[0122] 1. ELISA

[0123] (1) Antigen coating: B7-H3-2Ig and B7-H3-4Ig proteins were diluted to a concentration of 2 μg / mL with PBS (pH 7.4), and the coating volume was 50 μL / well. The cells were then placed in a 4°C refrigerator for 12 h.

[0124] (2) Blocking: Wash the plate three times with PBST, then add 3% BSA-PBST blocking solution to the wells and block at 37°C for 1 hour. Wash the plate three times.

[0125] (3) Add the sample to be tested: Add 100 μL of antibody diluted with PBS to each well, with a concentration of 20 μg / mL in the first well, followed by a 1:3 serial dilution for a total of 8 wells. Incubate at 37℃ for 2 h.

[0126] (4) Secondary antibody incubation: Use HRP-conjugated IgG secondary antibody, dilute the secondary antibody with 0.1% BSA-PBST (1:5000), 100 μL / well, and incubate at 37℃ for 1 h.

[0127] (5) Color development: Wash the plate 3 times, add TMB color development solution, 100 μL / well, and incubate at 37°C in the dark for 15 min.

[0128] (6) Termination: Add an equal volume (100 μL / well) of 1M hydrochloric acid solution to terminate the reaction. The reaction solution in the well changes from blue to yellow.

[0129] (7) Read the results: Measure the OD450 value using an ELISA reader within 15 minutes.

[0130] The binding of the A172-mu, A172-hu4 antibodies and the positive control antibody MGA271 shown in Example 1 was detected by ELISA, and the EC50 was calculated.

[0131] Table 1: EC50 values ​​(mean ± standard deviation) of A172 monoclonal antibody binding to B7-H3 protein before and after humanization.

[0132] The results showed that the affinity between A172-hu4 and B7H3 was not reduced, and was even better than that between A172-mu, indicating that the humanized A172-hu4 and B7H3 still have a strong affinity (Figure 2, Table 1).

[0133] 2. Flow cytometry

[0134] (1) Collect esophageal squamous cell carcinoma cells KYSE150, centrifuge, resuspend in buffer (PBS solution containing 2% FBS), count, and prepare a density of 2×10⁻⁶ cells / mL. 6 Add 200 μL of cell suspension to each EP tube, centrifuge at 2000 rpm for 5 min, and discard the supernatant.

[0135] (2) Dilute the antibody to be tested to different concentration gradients with buffer, starting with 30 μg / ml, then dilute 1:3. Take 200 μL of each concentration and add it to the above EP tube to resuspend the cells, so that the final antibody concentrations are 30, 10, 3, 1, 0.33, 0.11, 0.037, and 0.012 μg / mL. Use an EP tube with 200 μL of buffer added as a blank control.

[0136] (3) Incubate the EP tube on ice for 1 hour, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0137] (4) Use FITC-labeled IgG secondary antibody, diluted 1:200 with buffer. Resuspend cells in 200 μL of the diluted secondary antibody solution per tube. Incubate on ice in the dark for 1 h. Centrifuge and wash twice with PBS.

[0138] (5) Resuspend the cells in 200 μL of buffer, filter through a 300-mesh filter, and then detect the fluorescence intensity by flow cytometry.

[0139] The binding of A172-mu, A172-hu4, and MGA271 antibodies to the B7H3-positive esophageal cancer cell line KYSE150 was detected by flow cytometry, and the EC50 was calculated.

[0140] Table 2: EC50 values ​​(mean ± standard deviation) of A172 monoclonal antibody binding to B7-H3 before and after humanization.

[0141] The results showed that the affinity of A172-hu4 and B7H3 was not reduced and was superior to that of A172-mu and MGA271 antibodies, indicating that the humanized A172-hu4 and B7H3 still have a strong affinity (Figure 3 and Table 2).

[0142] 3. Surface plasmon resonance

[0143] (1) Capture A172-Mu and A172-Hu4 antibodies on the surface of ProteinA chip. Dilute the antibodies to 1 μg / mL with HBS-EP+ buffer and set the flow rate to 10 μL / min.

[0144] (2) Ten different concentration gradients of B7-H3 antigen were set up, and the antigen was diluted to 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM with 1×HBS-EP+ buffer. The flow rate of B7-H3 antigen was set to 30 μL / min, and the binding time was set to 300 s.

[0145] (3) Use Giycine 1.5 as the regeneration buffer and regenerate the chip according to the regeneration procedure.

[0146] (4) Analyze the results using GraphPad Prism 8 software, perform binding mode fitting, and calculate binding dynamic parameters.

[0147] The affinity of A172-mu, A172-hu4, MGA271 antibodies and B7H3 protein was detected using surface plasmon resonance (SPR) technology.

[0148] Table 3: KD values ​​of A172-hu4, A172-MU, MGA271 binding to B7-H3-4 Ig protein

[0149] Note: ka is the binding constant, kd is the dissociation constant, and KD is the equilibrium dissociation constant.

[0150] The results showed that the KD value of A172-Hu4 was 2.35 nM, the KD value of A172-mu was 2.84 nM, and the KD value of MGA271 was 1.45 nM. There was no significant difference in the KD values ​​between A172-hu4 and A172-mu, which were slightly higher than the positive control antibody MGA271, reaching the nanomolar level. This indicates that the affinity of the A172 antibody did not change significantly after humanization (Figure 4 and Table 3).

[0151] 4. Antibody-dependent cell-mediated cytotoxicity

[0152] (1) Collect cells in the logarithmic growth phase (target cells) and adjust the cell density to 1×10⁻⁶ cells using culture medium. 5 Cells per mL were seeded into 96-well plates, 100 μL per well, and cultured overnight until cells adhered.

[0153] (2) Dilute the antibody to 10 μg / mL with complete 1640 medium. Add 50 μL of the antibody to each well of a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 30 min.

[0154] (3) Collect cells, centrifuge, discard supernatant, and wash twice with PBS. Adjust cell density to 2 × 10⁶ cells using culture medium. 6 Add 100 μL of the above-mentioned 96-well plate (containing 200 ng / mL IL2) to each well (effect cell to target ratio of 20:1) after mixing well. Include wells for culture medium background, volume correction, target cell lysis, spontaneous release of target cells, and spontaneous release of effector cells. Incubate at 37°C in a 5% CO2 incubator for 18 h.

[0155] (4) Add 25 μL of the lysis buffer from the detection kit to the target cell lysis well and the volume correction well 45 min before detection.

[0156] (5) Centrifuge the 96-well plate at 300g for 3 min, and transfer 50 μL of the supernatant to another 96-well plate. Add 50 μL of LDH detection reagent to each well and incubate at room temperature in the dark for 20 min. Add 50 μL of the stop solution from the detection kit to each well. Measure the OD490 value using a microplate reader.

[0157] (6) Calculation: Calculate the cytotoxicity according to the formula: Cell cytotoxicity = [(experimental wells - culture medium background wells) - (target cell spontaneous release wells - culture medium background wells) - (effect cell spontaneous release wells - culture medium background wells)] / [(target cell lysis wells - culture medium correction wells) - (target cell spontaneous release wells - culture medium background wells)] × 100%.

[0158] The results showed that A172-hu4 had significant killing activity against AML cells THP-1 and U937, as well as ESCC cells KYSE70 and KYSE150, with statistically significant differences compared to the control group (P<0.01). The results are shown in Figures 5 and 6.

[0159] Example 3: Construction of CAR-T cells targeting B7H3

[0160] 1. Constructing a CAR vector targeting B7H3

[0161] The single-chain variable region fragments (scFv, as shown in SEQ ID NO:15 and 16, respectively) of the A172-hu4 and A172-mu antibodies shown in Example 1, and the single-chain variable region fragments (scFv, as shown in positions 22-266 of SEQ ID NO:42) of the A172-hu4 and A172-mu antibodies, were linked to the hinge region and transmembrane region fragments of CD8α (as shown in SEQ ID NO:17), the intracellular region of the co-stimulatory molecule 4-1BB (CD137) (as shown in SEQ ID NO:18), and the intracellular region of the CD3ζ chain (as shown in SEQ ID NO:19). The vector used was pCDH-CMV-MCS-EF1-copEGFP-T2A-PURO (Figure 7). The gene sequences were obtained from UniProt and NCBI websites. The CAR vector gene sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. The nucleotide sequences of A172-hu4-CAR, A172-mu-CAR and A172-hu3-CAR are shown in SEQ ID NO:32, 33 and 43, respectively.

[0162] 2. B7H3 CAR plasmid extraction

[0163] (1) Take the competent bacteria DH5α out of the -80℃ freezer and place it on ice to thaw.

[0164] (2) Take 1 μL of the light chain and heavy chain plasmid of the antibody and add them to an EP tube, then add 50 μL of DH5α to each tube. Incubate on ice for 30 min.

[0165] (3) Place the EP tube in a 42℃ water bath for 90 seconds and then quickly place it on ice for 2 minutes.

[0166] (4) Add 1 mL of LB liquid culture medium to the tube and place it in a shaker at 37°C with a shaking speed of 220 rpm for 1 h.

[0167] (5) Centrifuge the above bacterial solution to remove most of the supernatant, then mix the remaining bacterial solution and spread it on LB agar plates containing ampicillin, and incubate at 37°C for 12-16 h.

[0168] (6) The next day, single colonies were picked and placed in LB liquid medium containing ampicillin, and cultured at 37°C and 220 rpm for 12-16 hours with shaking.

[0169] (7) Extract plasmids according to the instructions of the large-volume plasmid extraction kit.

[0170] (8) Collect the plasmid solution, use Nanodrop to detect the plasmid concentration and check whether the absorbance ratios of A260 / A280 and A260 / A230 are within the normal range.

[0171] (9) Label the extracted plasmids and aliquot them, and store them at -20℃.

[0172] 3. Lentiviral packaging and concentration

[0173] (1) Prepare two centrifuge tubes, and add 5 mL of HEK293 medium to each tube. Add 200 μg of the target plasmid and packaging plasmid to one tube. Add 600 μL of PEI to the other tube. Incubate for 5 min.

[0174] (2) Filter the culture medium containing the plasmid into a new centrifuge tube using a 0.22 μm sterile filter. Slowly add the culture medium containing PEI to the filtered plasmid, mix thoroughly, and incubate together for 20 min.

[0175] (3) Transfer the plasmid and PEI mixture into a shake flask containing 293F cell suspension, and then add fresh culture medium to the shake flask up to 100 mL. Place the shake flask on a shaker in a 37°C, 5% CO2 incubator for incubation.

[0176] (4) After 6-8 hours, add 10% of the system's SMS293-SUPI feed and glutamine to the shake flask.

[0177] (5) After culturing for 48 hours, collect the virus solution. Take a small amount of the virus solution and observe under a fluorescence microscope whether the green fluorescence is greater than 90%. If it is greater than 90%, transfer the cell suspension into a 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, collect all the virus solution supernatant, filter it with a 0.45 μm sterile filter and transfer it into a sterile high-speed centrifuge tube. Add 3-4 mL of 20% sucrose to the bottom of the high-speed centrifuge tube and centrifuge at 4℃ and 20000g for 2 h.

[0178] (6) After centrifugation, discard the supernatant and resuspend the precipitate with a small amount of X-VIVO medium. The resuspended suspension is the concentrated virus solution. After aliquoting, store it in a -80℃ refrigerator.

[0179] 4. Lentiviral titer detection

[0180] Add 1×10 to each well of the 24-well plate 5Jurkat cells were infected, and 500 μL of fresh 1640 medium, virus solution, and 8 μg / mL polybrene were added to each well. Multiple concentrations of virus solution were prepared using serial dilutions, such as 8 μL, 2 μL, 0.5 μL, 0.125 μL, and 0.03125 μL, with a 4-fold serial dilution. Cells were incubated at 37°C in a 5% CO2 incubator. Fluorescence intensity was observed and detected by flow cytometry 48 h after infection. Transduction titer (TU / mL) = dilution factor × sample positivity rate × cell volume / virus volume (mL). The lentiviral titer of A172-hu4-CAR (as shown in SEQ ID NO:22) was 1.528 × 10⁻⁶. 8 / mL, the lentiviral titer of A172-mu-CAR (as shown in SEQ ID NO:21) is 1.25×10⁶ / mL. 9 / mL.

[0181] 5.CD3 + T lymphocyte isolation and culture

[0182] (1) Coat a six-well plate with CD3 monoclonal antibody at a final concentration of 2 μg / mL and RetroNection at a final concentration of 10 μg / mL, and place it in a 4°C refrigerator overnight or an incubator at 37°C for at least 5 hours.

[0183] (2) Draw 30 mL of peripheral blood from healthy volunteers into anticoagulant tubes containing heparin sodium and dilute the peripheral blood 1:1 with PBS. Add 4 mL of lymphocyte separation medium to each 15 mL centrifuge tube, and then slowly add 8 mL of diluted peripheral blood to the centrifuge tube. Centrifuge at 1500 rpm for 22 min at room temperature.

[0184] (3) After centrifugation, carefully aspirate the middle white membrane layer into a centrifuge tube, wash twice with PBS, and centrifuge at 1500 rpm for 10 min each time.

[0185] (4) Resuspend the cells in 20–50 μL of magnetic bead separation buffer, count them, and transfer them to EP tubes or centrifuge tubes, 1 × 10⁻⁶ per tube. 7 Each cell.

[0186] (5) Each 1×10 7 Add 80 μL of magnetic bead separation solution and 20 μL of CD3 beads to each cell. Mix thoroughly and incubate at 4°C in the dark for 15–30 min.

[0187] (6) After incubation, add 1 mL of magnetic bead separation buffer to the tube, gently mix, centrifuge at 1500 rpm for 10 min, wash once, discard the supernatant, resuspend the cell pellet in 500 μL of magnetic bead separation buffer, insert the magnetic column into the positive selection groove of the magnetic bead sorter, add the resuspended cells to the magnetic column, and rinse the magnetic column three times with magnetic bead separation buffer, adding 5 mL each time. At this time, the cells flowing through the magnetic column with the magnetic bead separation buffer are CD3 cells. - Cells, discard.

[0188] (7) Remove the magnetic column, add 1 mL of magnetic bead separation solution to the magnetic column, push the magnetic bead separation solution into a new centrifuge tube with the push handle, gently blow to mix, wash with 1 mL of 0.9% physiological saline, and centrifuge at 1500 rpm for 10 min.

[0189] (8) After washing, the cell pellet is the purified CD3. + T lymphocytes were resuspended in 1 mL of cell culture medium (X-VIVO medium + 1000 IU / mL IL-2) and counted.

[0190] (9)CD3 + T lymphocytes were counted at a rate of 2 × 10⁻⁶. 6 After inoculating the culture medium into each well of a 6-well plate and gently shaking to mix, add 2 mL of culture medium to each well and incubate overnight at 37°C in a 5% CO2 incubator.

[0191] 6. Lentiviral infection of human T cells

[0192] (1)CD3 + Two days after T cell activation, the cells were resuspended in centrifuge tubes and centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in 1 mL of fresh culture medium and counted. The cell density was adjusted, and the cells were seeded into the original six-well plates at 2 × 10⁶ cells per well. 6 Each cell.

[0193] (2) To CD3 + Add virus solution with an MOI of 10-15 and 2 μL of polybrene to T cells. MOI = virus titer × virus solution volume / cell number. Calculate the required volume of virus solution based on the virus titer. Mix the fresh culture medium, virus solution, and polybrene in a centrifuge tube beforehand, then add the mixture to the pre-coated CD3 cells. + T cells in a six-well plate.

[0194] (3) Place the six-well plate in a centrifuge and centrifuge at 1000g and 22℃ for 30min. After centrifugation, carefully and slowly place it into a 37℃, 5% CO2 incubator for overnight infection.

[0195] (4) Observe the cells in the 6-well plate that have been infected overnight, remove the viral supernatant, add 3 mL of fresh culture medium per well, and continue culturing.

[0196] (5) On the third day after infection, the T cells are basically fully activated and proliferate vigorously. At this time, the T cells are transferred into a culture flask, fresh culture medium is added, and culture continues.

[0197] Example 4: CAR-T cell transfection rate and CAR expression

[0198] (1) As shown in Example 3, B7H3 CAR virus transfects CD3 + About one week later, 2×10 T cells were collected. 5 After centrifugation of the transfected CAR-T cells, NT cells, and PBMC cells, the culture medium was discarded, 1 mL of PBS was added to resuspend the cell pellet and wash the cells, and the cells were centrifuged at 2000 rpm for 5 min at 4°C.

[0199] (2) After centrifugation, the supernatant was aspirated, and the cell pellet was resuspended in 200 μL of PBS containing 2% FBS in each tube. CAR-T cells were divided into two tubes: one tube was added with PE-B7H3 protein at a final concentration of 5 μg / ml, and the other tube was added with only 200 μL of PBS without protein incubation; NT cells were similarly added with only 200 μL of PBS without protein incubation; PBMCs were divided into three tubes: one tube was added with only 200 μL of PBS, one tube was added with PE-anti-human CD3 antibody at a final concentration of 5 μg / ml as a single-staining tube for PE staining, and the other tube was added with FITC-anti-human CD3 antibody at a final concentration of 5 μg / ml as a single-staining tube for FITC staining. After mixing, the EP tubes were incubated at 4℃ for 30 min.

[0200] (3) After incubation, centrifuge at 2000 rpm for 5 min at 4℃, discard the supernatant, add 500 μL of PBS containing 2% FBS to resuspend the cell pellet and wash twice, centrifuging at 2000 rpm for 5 min each time.

[0201] (4) Centrifuge, discard the supernatant, add 200 μL of PBS containing 2% FBS to resuspend the cell pellet, and filter the cell suspension into a tube through a 300-mesh sieve.

[0202] (5) Turn on the flow cytometer, first use PE-stained single-stain tubes and FITC-stained single-stain tubes to adjust the compensation size, and then use the instrument to detect the fluorescence intensity of each sample.

[0203] (6) The fluorescence intensity of each sample was analyzed and the transfection rate and CAR expression of CAR-T cells were calculated using FlowJo 10.0.7 software.

[0204] The results showed that the transfection rates of A172-mu, A172-hu3, and A172-hu4 CARs in HEK293 cells packaged with CAR virus, measured by flow cytometry, were 99.3%, 95.2%, and 98.9%, respectively, with CAR expression rates of 94.3%, 7.89%, and 94.3%, respectively. The CAR expression rate of A172-hu4 was significantly better than that of A172-hu3 (see Figure 8). The results showed that the infection efficiency of A172-hu4-CAR-T cells was 41%, with a CAR positivity rate of 31.5%; the infection efficiency of A172-mu-CAR-T cells was 46%, with a CAR positivity rate of 45.9% (see Figure 9).

[0205] Example 5: In vitro killing of tumor cells by A172-CAR-T cells

[0206] 1. Co-culture of A172-CAR-T cells with AML cells and ESCC cells

[0207] (1) Collect THP-1, U937 cells and KYSE70 and KYSE150 cells from the culture dish into centrifuge tubes, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0208] (2) Resuspend the cell pellet in 1 mL of fresh complete culture medium and count the cells, adjusting the cell suspension to 1 × 10⁻⁶. 5 / mL, extract 100μL and spread it into a 96-well plate.

[0209] (3) After the tumor cells are seeded, the 96-well plate is placed in an incubator at 37°C and 5% CO2 (adherent cells need to adhere to the wall before adding CAR-T cells, while suspension cells can be seeded directly into the well plate and then CAR-T cells are added). Then, CAR-T cells are prepared as described in Example 3.

[0210] (4) Collect the A172-CAR-T cells in the culture dish into a centrifuge tube, centrifuge at 1500 rpm for 10 min, and discard the supernatant.

[0211] (5) Resuspend the cell pellet in 1 mL of fresh complete culture medium and count the cells. Adjust the cell suspension to a suitable density and add A172-CAR-T to the target cell wells prepared in step (3) above in an appropriate ratio (AML cell line effector cells: target cells = 1:1, 2:1, 4:1, 8:1, ESCC cell line effector cells: target cells = 2:1, 4:1, 8:1). The final volume in the well is 200 μL.

[0212] (6) A172-CAR-T cells and target cells were co-cultured in an incubator at 37℃ and 5% CO2 for 18h.

[0213] 2. Detection of the killing rate of A172-CAR-T cells against tumor cells using the lactate dehydrogenase method.

[0214] (1) When A172-CAR-T cells and tumor cells are co-cultured, target cell release wells, target cell lysis wells, effector cell release wells, culture medium background wells, and volume correction wells are set up at the same time.

[0215] (2) After co-incubation for 18 hours, add 20 μL of lysis buffer to the target cell lysis well and incubate at 37°C and 5% CO2 for 45 minutes.

[0216] (3) After incubation, place the 96-well plate in a centrifuge at 300g for 3min, prepare a new 96-well plate, aspirate 50μL of the culture supernatant from each well into the new 96-well plate, add 50μL / well of LDH detection reagent to the supernatant, and incubate at room temperature in the dark for 20min.

[0217] (4) After incubation, add stop solution, 50 μL / well.

[0218] (5) Place the 96-well plate into an ELISA reader to detect the OD value at 490 nm.

[0219] (6) Calculate the cell killing rate according to the following formula to assess antibody-dependent cell-mediated cytotoxicity.

[0220] Cell cytotoxicity = [(experimental wells - culture medium background wells) - (target cell spontaneous release wells - culture medium background wells) - (effect cell spontaneous release wells - culture medium background wells)] / [(target cell lysis wells - culture medium correction wells) - (target cell spontaneous release wells - culture medium background wells)] × 100%. The results are shown in Figure 10.

[0221] 3. ELISA detection of IFN-γ and IL-2 levels in the supernatant after co-culturing A172-CAR-T cells and tumor cells.

[0222] (1) Draw the co-culture supernatant into a sterile EP tube, centrifuge at 2000 rpm for 20 min, and carefully extract the supernatant for use.

[0223] (2) Prepare 1× washing solution, detection antibody working solution and biotin-HRP working solution according to the instructions, and serially dilute the standard to several different concentrations.

[0224] (3) Calculate the number of wells required for the experiment, take out the corresponding number of strips, mark the position of each sample, and set 3 replicates for each sample, standard and blank.

[0225] (4) Add the standard, sample and diluent (added to the blank well) to the corresponding positions of the strip at a rate of 100 μL / well.

[0226] (5) Add 50 μL of detection antibody to each well, cover with sealing strip, and incubate at 200 rpm for 2 h on a horizontal shaker at room temperature.

[0227] (6) After the antibody incubation is complete, discard the liquid in the wells, add 300 μL of 1× washing buffer to each well, discard the liquid in the wells after 1 min, and invert the wells onto absorbent paper. Repeat 3 times.

[0228] (7) Add biotin-HRP at a rate of 100 μL / well, cover with sealing film, and incubate at room temperature on a horizontal shaker for 20 min.

[0229] (8) After biotin-HRP incubation, discard the liquid in the wells, add 300 μL of 1× washing solution to each well, discard the liquid in the wells after 1 min, and invert the wells onto absorbent paper. Repeat 3 times.

[0230] (9) Add 100 μL of TMB to each well and incubate at room temperature in the dark for 5–30 min. The reaction can be terminated based on the intensity of the color (dark blue) in the well. The recommended color development time is usually 10–20 min.

[0231] (10) Quickly add 100 μL of stop solution to each well to terminate the reaction.

[0232] (11) Within 10 minutes after the reaction is terminated, the plate is read and the readings are recorded simultaneously using a detection wavelength of 450 nm and a reference wavelength or correction wavelength of 610 nm.

[0233] (12) Plot a standard curve based on the concentration of the standard and its corresponding OD value. Then, calculate the cytokine concentration of each experimental well based on the standard curve equation.

[0234] The results showed that A172-CAR-T cells exhibited strong killing activity against AML cell lines THP-1 and U937, as well as ESCC cell lines KYSE70 and KYSE150, at different effector-to-target ratios. Among them, A172-hu4-CAR-T cells achieved a killing rate of about 40% against THP-1 and KYSE150 cells at an effector-to-target ratio of 2:1, which increased with the increase of the effector-to-target ratio. At an effector-to-target ratio of 8:1, the killing rate of A172-hu4-CAR-T cells against THP-1 and U937 cells was close to 100%, and the killing rate against KYSE70 and KYSE150 cells was about 80%, showing excellent killing function (see Figure 10).

[0235] 4. In vitro killing of tumor cells by A172-CAR-T cells

[0236] 4.1) Mouse model of disseminated AML xenograft

[0237] A mouse model of disseminated AML xenograft tumor was constructed using 6-8 week old female NOG mice by injecting THP-1-luc cells via the tail vein.

[0238] (1) Collect THP-1-luc cells in centrifuge tubes, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend in sterile PBS and count the cells. Adjust the cell density to 1×10⁻⁶ cells using PBS. 7 / mL, 100μL / mouse was injected into mice via the tail vein.

[0239] (2) One week later, weigh the mice and inject 1.2% afodin into the peritoneum at a dose of 25 mL / kg. After the mice are successfully anesthetized, inject the corresponding volume of 15 mg / mL fluorescein potassium salt into the peritoneum at a concentration of 10 μL / g body weight. After the potassium salt reaction takes ten minutes, place the mice in a small animal in vivo imaging instrument to observe the fluorescence content in the body.

[0240] (3) Based on the tumor cell burden of each mouse obtained through small animal imaging, the mice were randomly divided into four groups according to the fluorescence values: PBS group, NT group, A172-hu4-CAR-T group, and A172-mu-CAR-T group. The CAR-T cell density was adjusted to 5×10⁶ cells / mL using PBS. 7 / mL, each group was injected with 100μL of the corresponding T cells via tail vein, and the PBS group was injected with 100μL of sterile PBS per animal.

[0241] (4) After CAR-T cell injection, tumor burden was detected every 6 days using a small animal imaging system.

[0242] Observe the mice's mental state and health status daily, and record any abnormalities or deaths.

[0243] The mice were weighed and their weight recorded every 3 days.

[0244] On day 24, blood was collected from all mice via the orbital vein for routine blood tests. The mice were then euthanized, their organs were removed, weighed, and organ coefficients were calculated.

[0245] (5) Use GraphPad Prism 9.0 software to statistically analyze whether there were significant changes in body weight, tumor burden, blood routine and organ coefficients of mice in each treatment group during the treatment period.

[0246] The results showed that tumor cells in mice treated with A172-hu4-CAR-T cells and A172-mu-CAR-T cells were significantly inhibited and eliminated by day 7. There were no significant differences in any blood routine parameters or organ coefficients among the groups. This indicates that A172-CAR-T cells have good safety and efficacy in treating AML model mice (see Figure 11).

[0247] 4.2) ESCC xenograft mouse model

[0248] (1) Subcutaneous injection of tumor cells: KYSE150 cells were collected in centrifuge tubes, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in sterile PBS and counted. The cell density was adjusted to 1×10⁻⁶ cells using PBS. 7 100 μL / mouse was injected subcutaneously into the back of 6–8 week old female NOG mice.

[0249] (2) Monitor tumor growth daily until the tumor volume reaches 50-100 mm. 3 After lateral movement, the mice were divided into Z-shaped groups: PBS and NT groups had 4 mice per group, and A172-mu-CAR-T and A172-hu4-CAR-T groups had 5 mice per group.

[0250] (3) Each group was injected with 5×10 via the tail vein. 6 One CAR-T cell, 100 μL / corresponding T cell, and 100 μL / animal sterile PBS were injected into the PBS group.

[0251] (4) After CAR-T cell injection, the mental state and health status of the mice were observed daily. The tumor volume and body weight of the mice were measured every 3 days and recorded.

[0252] (5) When the mouse tumor reaches a size of 800 mm 3 All mice were euthanized, tumor tissue was removed, photographed, weighed, and then fixed in formalin.

[0253] (6) GraphPad Prism 9.0 software was used to statistically analyze whether the body weight of mice in each treatment group changed significantly during the treatment period, the tumor volume change curve, and the difference between the tumor weight of each group of mice and the control PBS group.

[0254] The results showed that the tumor volume and weight of mice in the A172-mu-CAR-T cell therapy group and the A172-hu4-CAR-T cell therapy group were smaller than those in the control group PBS and NT group, and they showed a significant inhibitory effect on esophageal squamous cell carcinoma in vivo (see Figure 12).

[0255] Sequence information

Claims

1. An isolated recombinant nucleic acid molecule encoding a chimeric antigen receptor (CAR) polypeptide, said CAR polypeptide comprising, from its N to C-terminus: a single-chain antibody (scFv) targeting a B7-H3 polypeptide, a transmembrane portion, and a cytoplasmic functional region, wherein The scFv contains light chain variable regions (VL) and heavy chain variable regions (VH) connected in any order: (a) The VL comprises the light chain CDR1 (CDR-L1) shown in SEQ ID NO:1, CDR-L2 shown in SEQ ID NO:2, and CDR-L3 shown in SEQ ID NO:3; and (b) The VH comprises the heavy chain CDR1 (CDR-H1) shown in SEQ ID NO:4, CDR-H2 shown in SEQ ID NO:5, and CDR-H3 shown in SEQ ID NO:6, and The cytoplasmic functional region comprises a 4-1BB cytoplasmic functional region and a CD3ζ cytoplasmic functional region connected in any order. Preferably, the amino acid sequence of the 4-1BB cytoplasmic functional region comprises or is composed of SEQ ID NO:18 and / or the amino acid sequence of the CD3ζ cytoplasmic functional region comprises or is composed of SEQ ID NO:

19. Preferably, the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 7 or 9, and / or the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10. Optionally, the VL and VH are connected by a linker comprising or is composed of the amino acid sequence shown in SEQ ID NO:

14. Preferably, the scFv comprises VL, a linker, and VH from the N to C ends. Optionally, the recombinant nucleic acid molecule further comprises a spacer region encoding a linker between the scFv and the transmembrane portion, and / or a nucleotide sequence encoding a signal peptide.

2. The recombinant nucleic acid molecule of claim 1, wherein: The scFv is composed of SEQ ID NO:15 or 16, preferably SEQ ID NO:16; and / or The transmembrane portion comprises a transmembrane portion selected from CD8 molecules, CD30 molecules, CD28 molecules, 4-1BB molecules, or CD3ζ molecules, for example, comprising or consisting of the amino acid sequence shown in SEQ ID NO:17; and / or The signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

13.

3. The recombinant nucleic acid molecule of claim 1 or 2, wherein the CAR polypeptide comprises the sequence shown in SEQ ID NO:21 or 22, or the sequence shown in amino acids 22-490 of SEQ ID NO:21 or 22, or the sequence shown in amino acids 22-490 of SEQ ID NO:21 or 22.

4. The recombinant nucleic acid molecule of any one of claims 1-3, comprising the sequence shown in SEQ ID NO:32 or 33, or the sequence shown in nucleotides 64-1473 of SEQ ID NO:32 or 33, or consisting of the sequence shown in SEQ ID NO:32 or 33, or the sequence shown in nucleotides 64-1473 of SEQ ID NO:32 or 33.

5. A recombinant vector comprising the nucleotide sequence of the recombinant nucleic acid molecule according to any one of claims 1-4.

6. A method of preparing a recombinant T lymphocyte, such as a CD3 + T lymphocyte, comprising transforming a T lymphocyte with the recombinant nucleic acid molecule of any one of claims 1-4, or the recombinant vector of claim 5.

7. A recombinant T lymphocyte, such as a recombinant CD3 + A T lymphocyte expressing a CAR polypeptide, wherein the CAR polypeptide is as defined in any one of claims 1 to 4, optionally, the recombinant T lymphocyte is obtained by the method of claim 6.

8. The use of the recombinant nucleic acid molecule according to any one of claims 1-4, or the recombinant vector according to claim 5, in the preparation of recombinant T lymphocytes.

9. The use of the recombinant nucleic acid molecule according to any one of claims 1-4, the recombinant vector according to claim 5, or the recombinant T lymphocyte according to claim 7 in the preparation of a medicament for treating cancers expressing the B7-H3 polypeptide, preferably the cancer being selected from leukemia such as acute myeloid leukemia, esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

10. A drug for treating cancers expressing the B7-H3 peptide, comprising: The recombinant nucleic acid molecule according to any one of claims 1-4, the recombinant vector according to claim 5, and / or the recombinant T lymphocyte according to claim 7, and a pharmaceutically acceptable vector. Preferably, the cancer is selected from leukemia such as acute myeloid leukemia, esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

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