Anti-B7-H3 monoclonal antibodies and their applications in cell therapy

Abstract

The present invention provides anti-B7-H3 monoclonal antibodies and their use in cell therapy. Specifically, the present invention provides scFv and antibodies that specifically target B7-H3, as well as specific CAR-T cells. The present invention also provides an engineered immune cell that can co-express a CAR targeting B7-H3 and a CAR or secreted protein targeting PD-L1, demonstrating excellent tumor killing efficacy.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 26, 2018, application number 201811125056.4, and invention name “Monoclonal antibodies against B7-H3 and their application in cell therapy”. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular, to anti-B7-H3 monoclonal antibodies and their application in cell therapy. Background Art

[0003] The targeting molecules expressed by tumor cells are the primary determinant of tumor identification and subsequent therapeutic application. Currently, various tumor treatment technologies, including targeted therapy, antibodies, and / or immunotherapies such as CAR-T cells, rely on specific targeting molecules expressed on tumors and their associated components. The specificity of targeting molecules is closely related to the efficacy and severity of tumor treatment side effects. B7-H3 is not expressed or is expressed at very low levels in normal tissues and cells, but is highly expressed on most solid malignant tumor cells and on vascular endothelial cells within tumors, making it a highly specific tumor targeting molecule.

[0004] Monoclonal antibodies are antibodies produced by a single B cell clone that target a specific antigenic epitope. By recognizing specific target molecules, monoclonal antibodies have been widely used in biological and medical research, clinical diagnosis, and treatment.

[0005] Chimeric antigen receptor T-cell therapy involves genetically modifying T cells with a chimeric antigen receptor (CAR) to create CAR-expressing T cells (CAR-T), which are then infused back into the body for therapeutic treatment. CAR-T cell therapy for tumors is a hot area of ​​current research and application development. Typically, CAR-T cells utilize the single-chain variable region (scFV) of the CAR molecule to recognize and kill specific targeting molecules on tumor cells, thereby exerting anti-tumor immune responses.

[0006] The efficacy of antibodies targeting B7-H3 in treating tumors is currently unclear. These antibodies are primarily used clinically to block and regulate inhibitory molecules on immune cells. However, due to the unclear immune function of B7-H3, their efficacy remains uncertain.

[0007] Currently, the efficacy of CAR-T cell therapy for solid tumors is suboptimal. Effective tumor-specific targeting molecules are a key factor in CAR-T cell therapy, but CAR-T cell therapy targeting solid tumors with currently known targeting molecules has yet to achieve definitive and lasting efficacy.

[0008] Therefore, there is an urgent need in this field to develop a class of antibodies that specifically target B7-H3 and have good tumor killing effects, as well as corresponding engineered immune cells. Summary of the Invention

[0009] The purpose of the present invention is to provide a class of antibodies that specifically target B7-H3 and have a good tumor-killing effect, and corresponding engineered immune cells.

[0010] The first aspect of the present invention provides an antibody heavy chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0011] CDR1 shown in SEQ ID NO: 1, 2 or 3,

[0012] CDR2 shown in SEQ ID NO: 4, 5 or 6, and

[0013] CDR3 shown in SEQ ID NO:7, 8 or 9.

[0014] In another preferred embodiment, the CDR of the heavy chain variable region comprises SEQ ID NO: N H ,N H +3, and N H +3 CDRs shown in 6, among which N H 1, 2 or 3 respectively.

[0015] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain B7-H3 binding affinity.

[0016] In another preferred embodiment, the heavy chain variable region further includes a human FR region or a mouse FR region.

[0017] In another preferred embodiment, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 19-24.

[0018] The second aspect of the present invention provides an antibody heavy chain, wherein the heavy chain has the heavy chain variable region as described in the first aspect of the present invention.

[0019] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0020] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin.

[0021] The third aspect of the present invention provides an antibody light chain variable region, wherein the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0022] CDR1' shown in SEQ ID NO: 10, 11 or 12,

[0023] CDR2' shown in SEQ ID NO: 13, 14 or 15, and

[0024] CDR3' shown in SEQ ID NO: 16, 17 or 18.

[0025] In another preferred embodiment, the CDR of the light chain variable region comprises SEQ ID NO: N L ,N L +3, and N L +3 CDRs shown in 6, among which N L 10, 11 or 12 respectively.

[0026] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain B7-H3 binding affinity.

[0027] In another preferred embodiment, the light chain variable region further includes a human FR region or a mouse FR region.

[0028] In another preferred embodiment, the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 25-30.

[0029] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain has the light chain variable region as described in the third aspect of the present invention.

[0030] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0031] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin.

[0032] A fifth aspect of the present invention provides an antibody having:

[0033] (1) the heavy chain variable region as described in the first aspect of the present invention; and / or

[0034] (2) The light chain variable region as described in the third aspect of the present invention.

[0035] In another preferred embodiment, the antibody has: the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0036] In another preferred embodiment, the affinity of the antibody for human B7-H3 (wild type) is Ka (1 / Ms) ≥ 1×10 5 (For example, 1×10 5 ~9×10 5 ), preferably ≥2×10 5 , preferably ≥2.5×10 5 .

[0037] In another preferred embodiment, the affinity of the antibody for human B7-H3 (wild type) is Kd (1 / s) ≤ 1.5×10 -3 (For example, 2×10 -5 ~1.5×10 -3 ), preferably ≤5×10 -4 , preferably ≤1×10 -4 .

[0038] In another preferred embodiment, the affinity of the antibody to human B7-H3 (wild type) is KD (M) ≤ 9 × 10 -9 (e.g. 1.0×10 -10 ~9×10 -9 ), preferably ≤5×10 -9 , preferably ≤1×10 -9 or ≤3×10 -10 In another preferred embodiment, the affinity of the antibody for human B7-H3 (preferably wild type) is Ka (1 / Ms) of 3.160E+5, Kd (1 / s) of 2.184E-4, and KD (M) of 6.913E-10.

[0039] In another preferred embodiment, the affinity of the antibody for human B7-H3 (preferably wild type) is Ka (1 / Ms) of 2.632E+5, Kd (1 / s) of 1.464E-3, and KD (M) of 5.563E-9. (Humanized HC1+LC1)

[0040] In another preferred embodiment, the antibody has an affinity for human B7-H3 (preferably wild type) of Ka (1 / Ms) of 1.842E+5, Kd (1 / s) of 1.034E-3, and KD (M) of 5.614E-9. (Humanized HC1+LC2)

[0041] In another preferred embodiment, the antibody has an affinity for human B7-H3 (preferably wild type) with a Ka (1 / Ms) of 3.272E+5, a Kd (1 / s) of 1.358E-3, and a KD (M) of 4.151E-9. (Humanized HC1+LC3)

[0042] In another preferred embodiment, the affinity of the antibody for human B7-H3 (preferably wild type) is Ka (1 / Ms) of 2.969E+5, Kd (1 / s) of 4.488E-5, and KD (M) of 1.512E-10. (Humanized HC2+LC1)

[0043] In another preferred embodiment, the antibody has an affinity for human B7-H3 (preferably wild type) of Ka (1 / Ms) of 3.240E+5, Kd (1 / s) of 3.582E-5, and KD (M) of 1.105E-10. (Humanized HC2+LC2)

[0044] In another preferred embodiment, the affinity of the antibody for human B7-H3 (preferably wild type) is Ka (1 / Ms) of 3.020E+5, Kd (1 / s) of 4.257E-5, and KD (M) of 1.410E-10. (Humanized HC2+LC3)

[0045] In another preferred embodiment, the antibody has an affinity for human B7-H3 (preferably wild type) of Ka (1 / Ms) of 2.923E+5, Kd (1 / s) of 6.360E-5, and KD (M) of 2.176E-10. (Humanized HC3+LC1)

[0046] In another preferred embodiment, the affinity of the antibody for human B7-H3 (preferably wild type) is Ka (1 / Ms) of 3.311E+5, Kd (1 / s) of 6.296E-5, and KD (M) of 1.901E-10. (Humanized HC3+LC2)

[0047] In another preferred embodiment, the antibody has an affinity for human B7-H3 (preferably wild type) of Ka (1 / Ms) of 5.036E+5, Kd (1 / s) of 6.333E-5, and KD (M) of 1.257E-10. (Humanized HC3+LC3)

[0048] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

[0049] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0050] In another preferred embodiment, the antibody is a monoclonal antibody.

[0051] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0052] In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in any one of SEQ ID NOs: 19-24; and / or

[0053] The light chain variable region sequence of the antibody is shown in any one of SEQ ID NOs: 25-30.

[0054] In another preferred embodiment, the antibody is of IgG type.

[0055] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0056] A sixth aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0057] (i) the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention; and

[0058] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0059] In another preferred embodiment, the tag sequence includes a 6His tag.

[0060] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0061] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0062] The seventh aspect of the present invention provides a CAR construct, wherein the antigen binding region of the CAR construct is a scFv that specifically binds to B7-H3, and the scFv has the heavy chain variable region as described in the first aspect of the present invention and the light chain variable region as described in the third aspect of the present invention.

[0063] In another preferred embodiment, the structure of the CAR is shown in Formula I below:

[0064] L-scFv-H-TM-C-CD3ζ-ZP (I)

[0065] Where,

[0066] Each "-" is independently a connecting peptide or a peptide bond;

[0067] L is none or a signal peptide sequence;

[0068] scFv is a single-chain variable region sequence targeting B7-H3;

[0069] H is none or hinge region;

[0070] TM is the transmembrane domain;

[0071] C is a co-stimulatory signal molecule;

[0072] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0073] Z is a coding sequence for no or self-cleaved protein;

[0074] P is absent or contains the coding sequence of PD1-CD28 or PD1-IL7R fusion protein.

[0075] In another preferred embodiment, the structure of the scFv is as shown in Formula A1 or A2:

[0076] V L -V H (A1); or

[0077] V H -V L (A2)

[0078] Among them, V L V is the light chain variable region of the anti-B7-H3 antibody; H is the heavy chain variable region of the anti-B7-H3 antibody; “-” is a connecting peptide (or flexible linker) or a peptide bond.

[0079] In another preferred embodiment, the structures of formula A1 and A2 are from N-terminus to C-terminus.

[0080] In another preferred embodiment, the V L and V H Connected by a flexible joint.

[0081] In another preferred embodiment, the flexible linker is 1-5 (preferably 2-4, more preferably 3-4) consecutive sequences represented by (G)4S.

[0082] In another preferred embodiment, the V L and V H Each is independently of murine, human, rabbit or human origin.

[0083] In another preferred embodiment, V L The amino acid sequence includes V selected from any one of SEQ ID No.: 25-30 L or its derivatives V L (or its active fragment).

[0084] In another preferred embodiment, V H The amino acid sequence includes V selected from any one of SEQ ID No.: 19-24 H or its derivatives V H (or its active fragment).

[0085] In another preferred embodiment, the scFv is a single-chain antibody variable region fragment of murine, human, human and murine chimeric, or fully humanized.

[0086] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CSF2RB, CD4, CD137, IL-2, IFNr, or a combination thereof.

[0087] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, CD80, CD86, or a combination thereof.

[0088] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0089] In another preferred embodiment, the C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0090] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from CD28 and / or 4-1BB (CD137).

[0091] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof.

[0092] In another preferred embodiment, the self-cleaving protein is selected from the following group: furin-V5-SGSG-T2A, furin-V5-SGSG-P2A, furin-V5-SGSG-E2A, furin-V5-SGSG-F2A, or a combination thereof.

[0093] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of furin-SGSG-T2A, furin-SGSG-P2A, furin-SGSG-E2A, furin-SGSG-F2A, or a combination thereof.

[0094] In another preferred embodiment, the structure of the fusion protein is as shown in Formula II:

[0095] L1-I1-L2-H1-TM1-C1 (II)

[0096] Where,

[0097] Each "-" is independently a connecting peptide or peptide bond

[0098] L1 is none or a signal peptide sequence;

[0099] I1 is the extracellular segment of PD-1;

[0100] L2 is absent or linked peptide element;

[0101] H1 is an optional hinge region;

[0102] TM1 is absent or transmembrane domain;

[0103] C1 is the absence or intracellular domain.

[0104] In another preferred embodiment, the L1 is a signal peptide of a protein selected from the group consisting of GM-CSF, CD4, CD8, IL-2, IFNr, TNF, or a combination thereof.

[0105] In another preferred embodiment, the signal peptide sequence of L1 is MALPVTALLLPLALLLHAARP (SEQ ID NO: 31).

[0106] In another preferred embodiment, the PD-1 extracellular segment has an amino acid sequence as shown in SEQ ID NO: 32.

[0107] In another preferred example, the amino acid sequence of the extracellular segment of PD-1 is shown in SEQ ID NO: 32.

[0108] In another preferred embodiment, the connecting peptide element is a sequence of 1-9 (preferably 2-7, more preferably 2-4) consecutive G4S, mlgG3UH, LFL, cTPRs, ZAG, β2m, polyPro (Glyc), polyPro, GlySer (Glyc), or a combination thereof.

[0109] In another preferred embodiment, the H1 is a hinge region of a protein selected from the group consisting of IgG4, CD8, CD28, CD137, or a combination thereof.

[0110] In another preferred embodiment, the TM1 is a transmembrane region of a protein selected from the group consisting of CD4, CD8, CD28, or a combination thereof.

[0111] In another preferred embodiment, the TM1 is a transmembrane region of a protein selected from the following group: CD28.

[0112] In another preferred embodiment, the TM1 has the amino acid sequence shown in SEQ ID NO: 33.

[0113] In another preferred embodiment, the amino acid sequence of TM1 is shown in SEQ ID NO: 33.

[0114] In another preferred embodiment, the C1 is an intracellular segment of a protein selected from the group consisting of CD137, CD28, IL-7R, or a combination thereof.

[0115] In another preferred embodiment, the C1 is an intracellular segment derived from CD28 and / or IL-7R.

[0116] In another preferred embodiment, the C1 has an amino acid sequence as shown in SEQ ID NO: 34.

[0117] In another preferred embodiment, the P element has the amino acid sequence shown in SEQ ID NO: 35.

[0118] In another preferred example, the coding sequence of the P element has a nucleotide sequence as shown in SEQ ID NO: 36.

[0119] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO: 37.

[0120] In another preferred example, the coding sequence of the CAR is shown in SEQ ID NO: 38.

[0121] An eighth aspect of the present invention provides an engineered immune cell, comprising:

[0122] (a) a first expression cassette, wherein the first expression cassette is used to express an exogenous CAR construct as described in the seventh aspect of the present invention; and

[0123] (b) optionally, a second expression cassette expressing a fusion protein comprising PD1-CD28 or PD1-IL7R.

[0124] In another preferred embodiment, the first expression cassette and the second expression cassette are connected (or connected in series) via a coding sequence of a self-cleaving protein.

[0125] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof.

[0126] In another preferred embodiment, the self-cleaving protein is selected from the following group: furin-V5-SGSG-T2A, furin-V5-SGSG-P2A, furin-V5-SGSG-E2A, furin-V5-SGSG-F2A, or a combination thereof.

[0127] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of furin-SGSG-T2A, furin-SGSG-P2A, furin-SGSG-E2A, furin-SGSG-F2A, or a combination thereof.

[0128] In another preferred embodiment, the result of the fusion protein is as shown in Formula II:

[0129] L1-I1-L2-H1-TM1-C1 (II)

[0130] Where,

[0131] Each "-" is independently a connecting peptide or peptide bond

[0132] L1 is none or a signal peptide sequence;

[0133] I1 is the extracellular segment of PD-1;

[0134] L2 is absent or linked peptide element;

[0135] H1 is an optional hinge region;

[0136] TM1 is absent or transmembrane domain;

[0137] C1 is the absence or intracellular domain.

[0138] In another preferred example, the fusion protein has the amino acid sequence shown in SEQ ID NO: 35.

[0139] In another preferred example, the coding sequence of the fusion protein has a nucleotide sequence as shown in SEQ ID NO: 36.

[0140] In another preferred embodiment, the first expression cassette contains a nucleic acid sequence encoding the CAR construct of claim 7.

[0141] In another preferred embodiment, the second expression cassette contains a nucleic acid sequence encoding the fusion protein.

[0142] In another preferred embodiment, the first expression cassette and the second expression cassette further comprise a promoter and / or a terminator, respectively.

[0143] In another preferred embodiment, the promoter is a mammalian promoter, preferably hEF1.

[0144] In another preferred embodiment, the sequence of the promoter is shown in SEQ ID NO: 39.

[0145] In another preferred embodiment, the first expression cassette and the second expression cassette are located on a vector or integrated into the chromosome of the engineered immune cell.

[0146] In another preferred embodiment, the first expression cassette and the second expression cassette are independent or connected.

[0147] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0148] In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

[0149] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, oncolytic viral vector, other gene transfer systems, or a combination thereof.

[0150] In another preferred embodiment, the vector is a lentiviral vector.

[0151] In another preferred embodiment, the vector is a transposon vector.

[0152] In another preferred embodiment, the cells are mammalian cells.

[0153] In another preferred embodiment, the immune cells are isolated from the body.

[0154] In another preferred embodiment, the immune cells are autologous.

[0155] In another preferred embodiment, the immune cells are non-autologous.

[0156] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0157] In another preferred embodiment, the immune cells are from primates (preferably humans).

[0158] In another preferred embodiment, the immune cells are selected from the following group:

[0159] (i) Chimeric antigen receptor T cells (CAR-T cells);

[0160] (ii) chimeric antigen receptor NK cells (CAR-NK cells); or

[0161] (iii) Exogenous T cell receptor (TCR) T cells (TCR-T cells)

[0162] In another preferred embodiment, the immune cells include: NK cells, T cells, NKT cells, (γδ) T cells, monocytes, or macrophages.

[0163] The ninth aspect of the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises:

[0164] (a) an antibody portion selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, or a combination thereof; and

[0165] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0166] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0167] In the tenth aspect, the present invention provides a heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the CAR construct according to the seventh aspect of the present invention, the immune cell according to the eighth aspect of the present invention, or the antibody-drug conjugate according to the ninth aspect of the present invention, for (i) preparing a drug or preparation for preventing and / or treating cancer or tumors; and / or (ii) preparing a detection reagent or kit.

[0168] In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0169] In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0170] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, osteosarcoma, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), nervous system tumor, glioma, neuroblastoma, metastatic malignant tumor, solid tumor abdominal metastasis, solid tumor pelvic metastasis, or a combination thereof.

[0171] In another preferred embodiment, the tumor expresses or highly expresses B7-H3.

[0172] In another preferred embodiment, the tumor includes a B7-H3 positive tumor.

[0173] In another preferred embodiment, the tumor includes a tumor that is positive for tumor vascular endothelial cells.

[0174] In another preferred embodiment, the antibody comprises an antibody in the form of a drug conjugate (ADC).

[0175] In another preferred embodiment, the detection reagent or kit is used to diagnose tumors that express or overexpress B7-H3.

[0176] In another preferred embodiment, the detection reagent or kit is used to detect B7-H3 protein in a sample.

[0177] In another preferred embodiment, the detection reagent is a detection sheet.

[0178] The eleventh aspect of the present invention provides a pharmaceutical composition, comprising:

[0179] The heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, the light chain described in the fourth aspect of the present invention, or the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, the CAR construct described in the seventh aspect of the present invention, the immune cell described in the eighth aspect of the present invention, and / or the antibody-drug conjugate described in the ninth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0180] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0181] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0182] In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1×10 3 -1×10 9 cells / ml, preferably 1×10 5 -1×10 8 cells / ml.

[0183] In another preferred embodiment, the pharmaceutical composition further contains other drugs that selectively kill tumor cells (such as nucleic acid drugs, antibody drugs, targeted drugs, other immune cell drugs, other CAR-T drugs, chemotherapy drugs, or a combination thereof).

[0184] The twelfth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0185] (1) the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention;

[0186] (2) the recombinant protein according to the sixth aspect of the present invention; and

[0187] (3) The CAR construct described in the seventh aspect of the present invention.

[0188] The thirteenth aspect of the present invention provides a vector, which contains the polynucleotide described in the twelfth aspect of the present invention.

[0189] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0190] The fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector described in the thirteenth aspect of the present invention or the polynucleotide described in the twelfth aspect of the present invention is integrated into its genome.

[0191] A fifteenth aspect of the present invention provides a method for preparing engineered immune cells, comprising the following steps:

[0192] (A) providing an immune cell to be modified; and

[0193] (B) introducing a first expression cassette and / or an optional second expression cassette into the immune cells to be modified, wherein the first expression cassette expresses the CAR construct as described in the seventh aspect of the present invention, and the second expression cassette expresses a fusion protein comprising PD1-CD28 or PD1-IL7R, thereby obtaining engineered immune cells.

[0194] In another preferred embodiment, in step (B), it includes (B1) introducing a first expression cassette expressing the CAR construct described in the seventh aspect of the present invention into the immune cell; and optionally, (B2) introducing a second expression cassette expressing the fusion protein into the immune cell; wherein the step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

[0195] In another preferred example, the first expression cassette contains a nucleic acid sequence encoding the CAR construct described in the seventh aspect of the present invention.

[0196] In another preferred embodiment, the second expression cassette contains a nucleic acid sequence encoding the fusion protein.

[0197] In another preferred embodiment, the first expression cassette and the second expression cassette are located on a vector or integrated into the chromosome of the engineered immune cell.

[0198] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0199] In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

[0200] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, oncolytic viral vector, other gene transfer systems, or a combination thereof.

[0201] In another preferred embodiment, the vector is a viral vector (such as a lentiviral vector).

[0202] In another preferred embodiment, the vector is a transposon vector.

[0203] In another preferred embodiment, the immune cells are T cells or NK cells.

[0204] In another preferred embodiment, the method further comprises the step of performing functional and effectiveness testing on the obtained engineered immune cells.

[0205] A sixteenth aspect of the present invention provides a method for detecting B7-H3 protein in a sample in vitro, the method comprising the steps of:

[0206] (1) contacting the sample with the antibody according to the fifth aspect of the present invention in vitro;

[0207] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of B7-H3 protein in the sample.

[0208] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0209] The seventeenth aspect of the present invention provides a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the fifth aspect of the present invention or the antibody-drug conjugate as described in the ninth aspect of the present invention.

[0210] The eighteenth aspect of the present invention provides a kit for preparing the engineered immune cells according to the eighth aspect of the present invention, characterized in that the kit comprises:

[0211] (a) a first container, and a first nucleic acid sequence located in the first container, wherein the first nucleic acid sequence comprises a first expression cassette for expressing the CAR construct of claim 7; and

[0212] Optionally, (b) a second container, and a second nucleic acid sequence located in the second container, wherein the second nucleic acid sequence comprises a second expression cassette for expressing the fusion protein.

[0213] In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

[0214] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0215] In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

[0216] In another preferred embodiment, the first and second nucleic acid sequences are located in the same vector.

[0217] A nineteenth aspect of the present invention provides a diagnostic kit comprising:

[0218] (1) a first container, wherein the first container contains the antibody according to the fifth aspect of the present invention; and / or

[0219] (2) A second container containing a secondary antibody against the antibody according to the fifth aspect of the present invention.

[0220] In another preferred embodiment, the kit contains the detection plate described in the seventeenth aspect of the present invention.

[0221] The twentieth aspect of the present invention provides a method for treating cancer or tumors, comprising administering to a subject in need thereof the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, the CAR construct described in the seventh aspect of the present invention, the immune cell described in the eighth aspect of the present invention, the antibody-drug conjugate described in the ninth aspect of the present invention, and the pharmaceutical composition described in the eleventh aspect of the present invention.

[0222] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0223] Figure 1 This study shows the expression of the B7-H3 membrane protein on various tumor cell types. Tumor cells were stained using a B7-H3 monoclonal antibody as the primary antibody and an APC-labeled anti-mIgG monoclonal antibody as the secondary antibody. B7-H3 expression on the membranes of various tumor cells was assessed by flow cytometry. Mouse IgG2a was used as the primary antibody isotype control.

[0224] Figure 2The results demonstrate that both the anti-human B7-H3 monoclonal antibody and its single-chain antibody protein have specific binding ability to B7-H3. CHO cells stably expressing B7-H3 were stained with the anti-human B7-H3 monoclonal antibody (antibody) and the anti-human B7-H3 single-chain antibody protein (Sc-FV), respectively. Flow cytometry analysis, using mouse IgG2a antibody (isotope) as an isotype control, demonstrated that both the anti-human B7-H3 monoclonal antibody and the anti-human B7-H3 single-chain antibody protein specifically bind to B7-H3.

[0225] Figure 3 The results demonstrate that the anti-human B7-H3 single-chain antibody protein and the B7-H3 monoclonal antibody have similar specific binding abilities for B7-H3. CHO cells stably expressing B7-H3 were stained with different concentrations of the anti-human B7-H3 monoclonal antibody and the anti-human B7-H3 single-chain antibody protein, respectively. Flow cytometry revealed that the anti-human B7-H3 monoclonal antibody and the anti-human B7-H3 single-chain antibody protein had similar specific binding abilities for B7-H3.

[0226] Figure 4 Schematic diagrams showing the construction of anti-B7-H3 chimeric antigen receptor or anti-B7-H3 chimeric antigen receptor co-expressed molecules.

[0227] They are: B7-H3 CAR-PDCA7R (BB-Z--PDCA7R), B7-H3 4-1BB-CAR (BB-Z), B7-H3 4-1BB-CAR co-expressing PD1-CD28 molecules (BB-Z–PD28), B7-H3 CD28-4-1BB-CAR (28BB-Z), and B7-H3 CAR lacking complete CD3ξ function (Del-Z).

[0228] Figure 5 The data show the cell proliferation response of CAR-T cells targeting B7-H3 with different structures to B7-H3 target cell antigen stimulation. B7-H3 CAR-PDCA7R T cells (BB-Z--PDCA7R) have the best proliferation capacity; B7-H3 4-1BB-CAR T cells (BB-Z), B7-H3 4-1BB-CAR T cells co-expressing PD1-CD28 molecules (BB-Z–PD28), and B7-H3CD28-4-1BB-CAR T cells (28BB-Z) also have good and similar proliferation capacity. As a control, B7-H3 CAR T cells lacking complete CD3ζ function (Del-Z) did not effectively proliferate due to lack of activation function.

[0229] Figure 6The cytokine secretion responses of B7-H3-targeting CAR-T cells with different constructs to target tumor cells are shown. CAR-T cells with different constructs specifically recognize and activate B7-H3-positive tumor cells, producing varying levels of cytokine secretion responses (TNF, IL-2, IFN-γ, IL-10, IL-6, and IL-4). MB231-H3KO, a breast cancer cell line with negative B7-H3 knockout, served as a negative control, and PBS served as a blank control.

[0230] Figure 7 The results show that CAR-T cells targeting B7-H3 with different structures can specifically kill target tumor cells in vitro. After co-incubation with target tumor cells at different effector-target ratios, the killing ability of CAR-T cells with different structures was tested. With the exception of B7-H3 CAR T cells lacking complete CD3ζ function (Del-Z), which lacked effective killing due to lack of activation function, the other CAR-T cells all had similar killing functions.

[0231] Figure 8 The study showed that CAR-T cells targeting B7-H3 with different structures can effectively inhibit the growth of breast cancer in animal models. In a mouse breast cancer model, intravenous injection of CAR-T cells targeting B7-H3 with different structures effectively inhibited tumor growth, except for the control B7-H3 CAR T cells lacking complete CD3ζ function (Del-Z).

[0232] Figure 9 The study showed that treatment with CAR-T cells targeting B7-H3 of different structures prolonged the survival of mice with lung metastasis cancer. In the lung metastasis cancer model, CAR-T cells targeting B7-H3 of different structures were intravenously injected. The results showed that mice treated with B7-H3 CAR T cells lacking complete CD3ζ function (Del-Z) as a control had the shortest survival, while B7-H3 CAR-PDCA7R T cells (BB-Z--PDCA7R), B7-H3 4-1BB-CAR T cells co-expressing PD1-CD28 molecules (BB-Z–PD28), and B7-H3CD28-4-1BB-CAR T cells (28BB-Z) all significantly prolonged the survival of mice.

[0233] Figure 10 The results show the efficacy of B7-H3 CAR-T cells in treating peritoneal implanted colorectal cancer. Luciferase-labeled LOVO cells were intraperitoneally injected into NCG mice (5×10 4LOVO cells / mouse) to establish a colorectal cancer peritoneal tumor model. Four days after tumor implantation, B7-H3 CAR-T cells were injected intraperitoneally for treatment, with 1.0×10 7 cells / mouse, as the treatment group; for homologous treatment with CD19 CAR-T cells, 1.0×10 7 Before treatment, 7 days after treatment, and 23 days after treatment, small animal imaging was performed to detect the fluorescence intensity of tumor cells to monitor tumor growth.

[0234] Figure 11 This image shows the killing effect of CAR-T cells constructed with humanized single-chain antibodies of varying affinity on target cells. Single-chain antibodies formed by combinations of different light and heavy chains after humanization have varying antibody affinities. CAR-T cells were constructed based on these single-chain antibodies with different affinities, including B7H3-CAR-T-ori (CAR-T constructed with unhumanized sequences), and CAR-T constructed with different combinations of humanized sequences: B7H3-CAR-T-h22-01, B7H3-CAR-T-h22-02, B7H3-CAR-T-h22-03, B7H3-CAR-T-h22-11, B7H3-CAR-T-h12-01, and B7H3-CAR-T-h21-01. These CAR-T cells were co-incubated with tumor target cells (B7H3-positive LOVO colorectal cancer cells and SKOV3 ovarian cancer cells) for 8 hours, and their killing effects (cell lysis rate) and cytokine secretion (IL-2, IFN-γ) were detected. The results showed that CAR-T cells derived from different single-chain antibody combinations had different killing effects on target cells and different levels of IL-2 and IFN-γ secretion after T cell activation. PBMC-only (untransfected T cells) served as a negative control; CD19-CAR-T cells and their targeted tumor cells, Nalm6 (CD19 positive, B7H3 negative), served as experimental controls for target specificity. DETAILED DESCRIPTION

[0235] Through extensive and in-depth research and extensive screening, the inventors unexpectedly developed for the first time a class of antibodies with high specificity and high affinity for B7-H3, as well as highly specific chimeric antigen receptor immune cells based on said antibodies. Specifically, the present invention unexpectedly obtained an anti-B7-H3 monoclonal antibody with extremely excellent affinity and specificity, and obtained a humanized antibody based on said antibody. The antibody of the present invention is capable of highly specific binding to the B7-H3 antigen, with very high affinity (its Ka (1 / Ms), Kd (1 / s) and KD (M) are all very excellent). The antibody of the present invention and its corresponding chimeric antigen receptor immune cell can specifically target B7-H3-positive or B7-H3-expressing or high-expressing tumor cells, and has excellent tumor-killing effect, while having no killing ability on normal cells. The present invention was completed on this basis.

[0236] the term

[0237] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0238] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0239] As used herein, a "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes within a cell.

[0240] As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

[0241] As used herein, "tumor antigen" refers to a biological molecule with antigenicity, the expression of which leads to cancer.

[0242] As used herein, "single-chain variable region fragment (ScFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for modifying ScFv are known to those skilled in the art.

[0243] As used herein, the terms "administer" and "treat" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer" and "treat" can refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, or contact of a fluid with a cell. "Administer" and "treat" also refer to in vitro and ex vivo treatment with an agent, diagnostic, binding composition, or with another cell. "Treatment," when applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; and includes contact of an anti-human LAG-3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0244] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any of the anti-human B7-H3 antibodies and compositions of the present invention, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0245] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0246] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0247] B7-H3

[0248] The human B7-H3 molecule, a member of the B7 family, is a type I transmembrane protein containing 506 amino acids and a molecular weight of 110 kDa. The human B7-H3 gene was first cloned from a cDNA library derived from human dendritic cells. Because of its structural similarity to B7 family genes, it was named B7 Homolog 3, or B7-H3 for short. The B7-H3 protein is a type I transmembrane protein with extracellular IgC- and IgV-like domains and an intracellular highly variable signaling domain, directed by a signal peptide. Its amino acid sequence shares 20% to 27% homology with other members of the B7 family.

[0249] When B7-H3 was first discovered, research focused on immunology. However, different studies have reported that B-H3 has both positive co-stimulatory activation and immunosuppressive functions on T cells, so its function is still unclear. Some studies have reported that B7-H3 molecules can co-stimulate CD4 + T cells and CD8 + T cell proliferation, enhanced induction of T cell immune killing response, selective stimulation of the secretion of IFN-γ, IL-8, TNF-α and IL-10, etc. Subsequent studies have shown that B7-H3 can negatively regulate T cell activation and inhibit CD4 + T cell activation and the secretion of corresponding cytokines such as IFN-γ and IL-4. At the same time, B7-H3 may also participate in the function of Treg to inhibit DC activation and antigen presentation.

[0250] The B7-H3 protein is not expressed or is expressed at very low levels in normal tissues and cells, but is highly expressed in various tumor tissues and is associated with tumor progression, poor patient prognosis, and poor clinical outcomes. Various studies have found B7-H3 expression in various tumor tissues, including non-small cell lung cancer, prostate cancer, melanoma, breast cancer, and pancreatic cancer. Furthermore, high B7-H3 expression is positively correlated with lymph node or bone metastasis, treatment resistance, postoperative progression and recurrence, and patient mortality. Therefore, B7-H3 may serve as a novel solid tumor targeting molecule.

[0251] PD-1 extracellular domain

[0252] The PD-1 extracellular segment is the extracellular segment of the PD-1 molecule:

[0253] PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH(SEQ IDNO:32)

[0254] Antibody

[0255] As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and order of their constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Igs are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains, depending on the constant region. Each of the five classes of Ig can have either kappa or lambda chains. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0256] The antibody light chain of the present invention may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0257] In the present invention, the antibody heavy chain described in the present invention may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is the constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0258] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred. The term "murine antibody" as used herein refers to a monoclonal antibody against human B7-H3 prepared according to the knowledge and skill in the art. During preparation, a test subject is injected with a B7-H3 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine B7-H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or variants thereof.

[0259] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody.

[0260] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. In order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0261] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., B7-H3). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include

[0262] (i) Fab fragment, consisting of V L 、V H , CL and CH1 domains;

[0263] (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0264] (iii) By V H and the Fd fragment consisting of the CH1 domain;

[0265] (iv) V of a single arm of the antibody H and V L Fv fragment composed of structural domains.

[0266] Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. H and V L The polypeptide linker between the domains can form the structure required for antigen binding.

[0267] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0268] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a B7-H3 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation.

[0269] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 1O -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0270] The term "competitive binding" refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of human B7-H3 as the monoclonal antibody of the present invention and binds to the antigen. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human B7-H3 recognized by the monoclonal antibody of the present invention.

[0271] The term "KD" or "Kd" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the invention exhibit dissociation equilibrium constants of less than about 10 -7 M, for example, less than about 10 -8 M, 1O -9 M or 10 -10 The dissociation equilibrium constant (KD) of 5 M or less binds to B7-H3, as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.

[0272] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0273] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0274] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

[0275] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0276] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0277] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0278] Table A

[0279] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0280] Human B7-H3 specific antibody

[0281] The present invention provides an anti-human B7-H3 antibody (hereinafter referred to as B7-H3 antibody). Specifically, the present invention provides a high-specificity and high-affinity antibody against B7-H3, which comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (V H ) amino acid sequence, the light chain contains a light chain variable region (V L ) amino acid sequence. The B7-H3 antibody of the present invention stimulates antigen-specific T cell responses, enhancing the anti-tumor effect of T cells, thereby maximizing the patient's own immune system response to tumors and achieving the purpose of killing tumor cells.

[0282] Preferably, the heavy chain variable region (V H ) amino acid sequence and light chain variable region (V L ) amino acid sequence is selected from the group consisting of:

[0283] a1) SEQ ID NO: 1;

[0284] a2) SEQ ID NO: 2;

[0285] a3) SEQ ID NO: 3;

[0286] a4) SEQ ID NO: 4;

[0287] a5) SEQ ID NO: 5;

[0288] a6) SEQ ID NO: 6;

[0289] a7) SEQ ID NO: 7;

[0290] a8) SEQ ID NO: 8;

[0291] a9) SEQ ID NO: 9;

[0292] a10) SEQ ID NO: 10;

[0293] a11) SEQ ID NO: 11;

[0294] a12) SEQ ID NO: 12;

[0295] a13) SEQ ID NO: 13;

[0296] a14) SEQ ID NO: 14;

[0297] a15) SEQ ID NO: 15;

[0298] a16) SEQ ID NO: 16;

[0299] a17) SEQ ID NO: 17;

[0300] a18) SEQ ID NO: 18

[0301] Any of the above amino acid sequences has a B7-H3 binding affinity after adding, deleting, modifying and / or replacing at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0302] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0303] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0304] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG, IgM antibodies or other subtypes of antibodies.

[0305] The animal is preferably a mammal, such as a mouse.

[0306] The antibodies of the present invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies targeting human B7-H3.

[0307] In a preferred embodiment of the present invention, any one or more of the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 9, or sequences thereof having B7-H3 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, is located in the heavy chain variable region (V H )'s CDR region.

[0308] In a preferred embodiment of the present invention, any one or more of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17 and 18, or sequences thereof having B7-H3 binding affinity after addition, deletion, modification and / or substitution of at least one amino acid, is located in the light chain variable region (V L )'s CDR region.

[0309] In a more preferred embodiment of the present invention, V H CDR1, CDR2, and CDR3 are independently selected from any one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9, or sequences thereof having B7-H3 binding affinity after addition, deletion, modification, and / or substitution of at least one amino acid; V L CDR1, CDR2, and CDR3 are independently selected from any one or several sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, and 18, or sequences thereof having B7-H3 binding affinity after addition, deletion, modification, and / or substitution of at least one amino acid.

[0310] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0311] In the present invention, the number of amino acids added, deleted, modified and / or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0312] Antibody preparation

[0313] Any method suitable for producing monoclonal antibodies can be used to produce the B7-H3 antibodies of the present invention. For example, animals can be immunized with linked or naturally occurring B7-H3 proteins or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration.

[0314] Any suitable form of B7-H3 can be used as an immunogen (antigen) to generate non-human antibodies specific for B7-H3 and screen the biological activity of the antibodies. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0315] Exemplary methods for producing the B7-H3 antibodies of the present invention are described in Example 1.

[0316] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In the present invention, the antibody is an IgG antibody, and IgG1 or IgG4 subtype is used.

[0317] Likewise, any type of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains, or variants thereof can be used in the compounds and methods of the invention.

[0318] Exemplary methods for humanizing the B7-H3 antibodies of the present invention are described in Example 1.

[0319] The DNA sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of different light and heavy chains can be fused together in various combinations to form single-chain antibodies. Optimized single-chain antibodies can be obtained by testing and analyzing the functions of single-chain antibodies with different combinations or linkage modifications.

[0320] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0321] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0322] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0323] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

[0324] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0325] The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

[0326] The steps of transforming host cells with recombinant DNA described in the present invention can be carried out using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, conventional culture medium is used under appropriate conditions.

[0327] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0328] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0329] Chimeric Antigen Receptor (CAR)

[0330] Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition domain, typically a single-chain variable fragment (scFv), a transmembrane region, and an intracellular costimulatory signaling domain. The design of CARs has evolved through the following process: First-generation CARs contain only a single intracellular signaling component, CD3ζ or FcγRI. Because they contain only a single activation domain, they induce only transient T cell proliferation and minimal cytokine secretion, but lack the ability to provide long-term T cell proliferation signals or sustained anti-tumor effects in vivo, resulting in limited clinical efficacy. Second-generation CARs incorporate a costimulatory molecule, such as CD28, 4-1BB, OX40, or ICOS, into the existing structure. Compared to first-generation CARs, these CARs significantly enhance the persistence of CAR-T cells and their ability to kill tumor cells. New immune costimulatory molecules, such as CD27 and CD134, have been added in tandem to these second-generation CARs, resulting in the development of third- and fourth-generation CARs.

[0331] The extracellular segment of CARs can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity, and secretion of cytokines, thereby eliminating the target cells. First, the patient's autologous cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. This method has a very low probability of developing graft-versus-host disease, and the antigen is recognized by the immune cells in a non-MHC restricted manner.

[0332] CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies, a high response rate that has not been achieved by any previous treatment method, and has triggered a wave of clinical research around the world.

[0333] Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and / or a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0334] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0335] The CAR of the present invention, when expressed in T cells, can perform antigen recognition based on antigen binding specificity. When it is combined with its associated antigen, it affects tumor cells, causing tumor cells to not grow, be prompted to die or otherwise be affected, and causes the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from costimulatory molecules and / or ζ chains. Preferably, the antigen binding domain is fused with the intracellular domain of a combination of a 4-1BB signaling domain and / or a CD3ζ signaling domain.

[0336] As used herein, "antigen binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments that have antigen binding activity. Fv antibodies contain the variable regions of the heavy and light chains of antibodies, but no constant regions, and are the smallest antibody fragments that have all the antigen binding sites. Generally, Fv antibodies also contain V H and V L The polypeptide linker between the domains is capable of forming the structure required for antigen binding. The antigen-binding domain is typically a scFv (single-chain variable fragment). The size of an scFv is generally one-sixth the size of a complete antibody. A single-chain antibody is preferably a single amino acid sequence encoded by a single nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes the highly expressed tumor antigen B7-H3, preferably a single-chain antibody.

[0337] In the present invention, the scFv of the present invention also includes conservative variants thereof, which refers to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the scFv of the present invention.

[0338] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0339] In the present invention, the number of amino acids added, deleted, modified and / or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0340] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0341] In the present invention, the CAR of the present invention is as described in the seventh aspect of the present invention.

[0342] Chimeric antigen receptor T cells (CAR-T cells)

[0343] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" all refer to the CAR-T cells described in the seventh aspect of the present invention. The CAR-T cells of the present invention can target tumor antigens (such as B7-H3).

[0344] After expression, the CAR of the present invention will pass through the cell membrane and be localized on the cell membrane.

[0345] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction for a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0346] Chimeric antigen receptor NK cells (CAR-NK cells)

[0347] As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell of the first aspect of the present invention. The CAR-NK cell of the present invention can target tumor antigens (such as B7-H3).

[0348] Natural killer (NK) cells are a major type of immune effector cell that protects the body from viral infection and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and possess enhanced anti-tumor cytotoxicity.

[0349] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages: (1) they directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0350] exogenous T cell antigen receptor

[0351] As used herein, an exogenous T cell antigen receptor (T cell receptor, TCR) is a TCR whose α chain and β chain are cloned from tumor-reactive T cells by gene transfer technology, and is exogenously transferred into T cells by genetic engineering using a lentivirus or retrovirus as a vector.

[0352] T cells modified with exogenous TCR can specifically recognize and kill tumor cells, and by optimizing the affinity of TCR with tumor-specific antigens, the affinity of T cells with tumors can be increased, thereby improving the anti-tumor effect.

[0353] carrier

[0354] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0355] The present invention also provides vectors into which the expression cassettes of the present invention are inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0356] In brief summary, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0357] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0358] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0359] Further, expression vector can be provided to cell in the form of viral vector.Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism and work in the origin of replication, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0360] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0361] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0362] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto for high-level expression. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0363] In order to evaluate the expression of the CAR polypeptide or its portion, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0364] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is following gene: it is not present in receptor organism or tissue or is expressed by receptor organism or tissue, and its coded polypeptide, the expression of this polypeptide is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter gene is measured under the appropriate time. Suitable reporter gene can comprise the gene (for example, Ui-Tei etc., 2000FEBS Letters479:79-82) of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein. Suitable expression system is well known and can utilize known technology to prepare or obtain commercially. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as promoter. Such promoter region can be connected to reporter gene and be used to evaluate the ability of reagent regulating promoter-driven transcription.

[0365] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0366] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0367] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0368] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0369] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0370] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0371] preparation

[0372] The present invention provides a composition comprising the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, the antibody of the fifth aspect, the recombinant protein of the sixth aspect of the present invention, the CAR construct of the seventh aspect of the present invention, the immune cell of the eighth aspect of the present invention, or the antibody-drug conjugate of the ninth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 9 cells / ml, preferably 1×10 5 -1×10 8 cells / ml.

[0373] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0374] Therapeutic applications

[0375] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the present invention. The transduced T cells can target the tumor cell marker B7-H3 protein, synergistically activate T cells, and elicit a cellular immune response, thereby significantly enhancing their efficiency in killing tumor cells from malignant tumors.

[0376] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of administering the CAR-T cells of the present invention to the mammal.

[0377] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach significantly reduces the risk of graft-versus-host disease, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0378] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain for an extended period of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, B7-H3 CAR-T cells induce a specific immune response against cells expressing B7-H3.

[0379] Although the data disclosed herein specifically disclose a lentiviral vector comprising an anti-B7-H3 scFv, hinge and transmembrane regions, and CD28 and / or 4-1BB (CD137), and a CD3ζ signaling domain, and optionally a coding sequence for a self-cleaving protein and optionally a coding sequence for a PD1-CD28 or PD1-IL7R fusion protein, the present invention should be construed to include any number of variations to each of the construct components.

[0380] Treatable cancers include tumors that are not vascularized or substantially not yet vascularized, and vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancer treated with the CAR of the present invention include but are not limited to cancer, blastoma and sarcoma, and certain leukemia or lymphoma, benign and malignant tumors and malignant tumors, such as sarcoma, cancer and melanoma. Also included are adult tumors / cancers and childhood tumors / cancers.

[0381] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0382] Solid tumor is the abnormal mass of the tissue that does not usually comprise cyst or liquid zone.Solid tumor can be benign or malignant.Dissimilar solid tumors are named with the cell type that forms them (such as sarcoma, cancer and lymphoma).Solid tumor such as sarcoma and cancer example comprises gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, osteosarcoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), nervous system tumor, glioma, neuroblastoma, metastasis row malignant tumor, solid tumor abdominal cavity metastasis, solid tumor pelvic cavity metastasis.

[0383] The CAR-T cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0384] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0385] In vitro procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0386] In addition to the use of 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 an antigen in a patient.

[0387] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of CAR-modified T cells of the present invention to a subject in need thereof.

[0388] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0389] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0390] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 3 to 10 8 The dose of cells / kg body weight is preferably 10 5 to 10 6The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (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 easily determined by a person skilled in the art of medicine by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0391] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0392] In certain embodiments of the present 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 (e.g., before, simultaneously, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present 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 compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0393] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 3 to 1×10 9The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0394] Detection Uses and Kits

[0395] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0396] In the present invention, the samples used include cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0397] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0398] The present invention also provides a kit comprising the antibody (or fragment thereof) and scFV of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0399] The main advantages of the present invention include:

[0400] (1) The antibodies of the present invention have the characteristics of high affinity and high specificity.

[0401] (2) The humanized antibody or scFv of the present invention still has high affinity and high specificity for B7-H3.

[0402] (3) The engineered immune cells of the present invention can target tumor antigens (such as B7-H3) and thereby selectively kill tumor cells.

[0403] (4) The engineered immune cells of the present invention can target the proliferating vascular endothelial cells within the tumor, inhibit or damage tumor cells by destroying the tumor's angiogenesis and blood supply, thereby simultaneously targeting tumor cells and tumor blood vessels, and killing tumor cells more effectively.

[0404] (5) A type of engineered immune cells of the present invention can co-express CAR targeting B7-H3 and CAR targeting PD-L1 or secreted proteins, thereby enhancing the killing effect on tumor cells.

[0405] (6) The CAR targeting B7-H3 and the fusion protein targeting PD-L1 in the present invention have a synergistic effect, which can improve the activation, proliferation, cytokine secretion and migration of CAR-T cells, improve the killing function of CAR-T cells in the body, promote the migration and homing of CAR-T cells to tumor tissues, increase the retention time of CAR-T cells in the body, and enhance the ability to form memory cells. Therefore, compared with a single CAR, it can enhance the therapeutic effect of CAR-T cells, especially improve the effect of CAR-T cells in treating solid tumors.

[0406] (7) The present invention is the first to use the single-chain antibody variable region (scFV) derived from the B7-H3 monoclonal antibody to construct B7-H3-specific CAR-T cells (B7-H3-CAR-T), and to verify the function of B7-H3-CAR-T cells in vitro and in vivo in animal models, as well as their therapeutic efficacy against various tumors.

[0407] (8) The monoclonal antibody against B7-H3 of the present invention can be further applied to bispecific antibodies, ADC antibodies, biological reagents, clinical diagnostic reagents, imaging reagents, etc.

[0408] (9) B7-H3 CAR cells and their novel CAR structures and technologies can not only be used to form CAR-T cells from T cells, but can also be used to genetically modify and improve other immune cells, such as NK cells.

[0409] (10) The novel CAR-T structure and technology of the present invention can be used for research and development and application in combination with other targeting molecules.

[0410] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0411] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0412] Example 1 Preparation of Antibodies

[0413] Following conventional methods, mice were immunized with B7-H3 protein, and serum was harvested to verify its affinity for B7-H3. Mouse spleen cells were then harvested to prepare and screen monoclonal antibody hybridoma cells. Total RNA from the monoclonal antibody hybridoma cells was extracted and purified. Monoclonal antibody cDNA was obtained through reverse transcription of the RNA and 5'-RACE-cDNA amplification. Using this cDNA as a template, PCR was used to amplify the cDNA sequences of the light and heavy chain variable regions of the monoclonal antibody. The cDNA sequences of the light and heavy chain variable regions were then linked to a plasmid vector using TA vector cloning technology.

[0414] Example 2 Determination of Antibody Sequences

[0415] The heavy chain and light chain plasmids are transformed into competent bacteria, and single clones are screened on cloning plates.

[0416] Plasmids were extracted and purified from monoclonal colonies, and the cDNA sequences of the light and heavy chain variable regions were obtained by conventional DNA sequencing.

[0417] The antibody variable region sequencing results were compared with the Kabat database to interpret the CDR region and select the correct antibody sequence.

[0418] The single-chain antibody fragment (scFv) was obtained by connecting the heavy chain and light chain variable region sequences through a short polypeptide linker and ligated to the phIgV and pmIgV plasmid vectors.

[0419] The plasmid vector was transfected into 293T cells to regain the single-chain antibody protein, and then the function of the single-chain antibody protein was verified.

[0420] Example 3B7-H3 is expressed in various types of tumor cells

[0421] Paraffin sections of various tumors were immunohistochemically stained using conventional methods: antigen retrieval using a citric acid / microwave steam bath at 100°C; staining was performed using a B7-H3 mAb primary antibody, an ABC complex kit, and Streptavidin-HRP. Microscopic observation revealed that B7-H3 is highly expressed in a variety of solid tumors, including colorectal, ovarian, breast, gastric, liver, pancreatic, prostate, malignant gliomas, neuroblastomas, head and neck tumors, and malignant melanoma. Furthermore, B7-H3 is specifically expressed in proliferating vascular endothelial cells within solid tumors.

[0422] B7-H3 mAb was used to measure B7-H3 expression in various tumor cell lines using conventional flow cytometry. The results showed that B7-H3 was highly expressed in multiple solid tumor cell lines.

[0423] Example 4 Identification of the Binding Ability of the Single-Chain Antibody scFV of B7-H3

[0424] After binding of labeled B7-H3 single-chain antibody or its scFv protein to CHO cells expressing human B7-H3, affinity was tested by flow cytometry. Under the same conditions, affinity neutralization tests were performed with the addition of varying concentrations of B7-H3 protein to verify binding specificity. The binding abilities of the monoclonal antibody and its scFv protein to human B7-H3 were compared at varying concentrations.

[0425] The results showed that the B7-H3 monoclonal antibody and its single-chain antibody scFV have equivalent binding abilities to B7-H3 and can effectively and specifically target the B7-H3 molecule.

[0426] Example 5 B7-H3 CAR and its co-expression with different molecules to form new CAR-T cells

[0427] Using overlap extension PCR, a single-chain antibody fragment (scFv) against human B7-H3, the CD8 hinge and transmembrane regions, the intracellular signaling domains of CD28 and / or 4-1BB, and the activation domain of CD3ζ were linked to construct the CAR cDNA construct. Furthermore, the extracellular fragment of PD-1 and the intracellular domain of CD28, and the extracellular fragment of PD-1 and the intracellular functional mutant receptor fragment of IL-7 were linked to construct fusion cDNAs of PD1-CD28 (PD28) and PDCA7R, respectively, using overlap extension PCR. Using the Fu-V5-SGSG-T2A sequence as the splicing linker, the CAR cDNA was linked to the PD28, PDCA7R, CD28, and / or EGFP fragments to construct the gene constructs for co-expressing CAR molecules.

[0428] Typical CAR structures are as follows Figure 4 As shown, the amino acid sequence of the CAR structure containing the co-expression molecule is shown in SEQ ID NO.: 37, and the nucleotide sequence encoding the CAR structure containing the co-expression molecule is shown in SEQ ID NO.: 38.

[0429] Using molecular cloning techniques, the CAR or co-expressed CAR construct is linked to a lentiviral expression vector. Second- or third-generation lentivirus is packaged and prepared in Lenti-X 293T cells or 293T cells using conventional PEI or calcium phosphate precipitation methods. The resulting lentiviral stock is filtered through a 0.45μm filter, concentrated by centrifugation, titered, and quickly frozen in liquid nitrogen before storage at -80°C.

[0430] The virus is incubated with a diluted fibronectin-coated culture plate to allow viral particles to adhere to the plate. Activated PBMCs or T cells are then seeded into the culture plate along with T cell culture medium containing IL-2. After centrifugation at 800g for 1 hour, the plate is placed in an incubator for viral transfection. After 48 hours of culture, the expression and phenotype of the CAR and co-expressed molecules are assayed. Subsequently, the CAR-T cells are cultured in a medium containing cytokines such as IL-2, IL-7, and IL-15 until harvest.

[0431] Example 6 CAR-T cells respond to target cell antigen stimulation

[0432] Freshly infected CAR-T cells were replaced with fresh medium and stimulated with irradiated target cells at a 1:1 effector-target ratio for 3 days. Cells were then counted using trypan blue staining. Three rounds of stimulation were repeated, each lasting 5-7 days. No exogenous cytokines were added, and only half of the medium was replaced. The total number of CAR-T cells was counted after each round.

[0433] B7-H3 CAR-T cells of various structures can effectively proliferate after being stimulated by B7-H3 antigens. At the same time, B7-H3 CAR-T cells co-expressing PDCA7R (B7-H3 CAR-CARPDCA7R T cells) have greater advantages in maintaining cell stability and proliferation ability after antigen-specific stimulation.

[0434] Example 7 Cytokine secretion levels after co-culture of CAR-T cells and tumor cells

[0435] After B7-H3 CAR-T cells with various structures were co-incubated with target tumor cells irradiated or not with 100 Gy, the secretion of various cytokines was detected and analyzed by flow cytometry using a CBA kit.

[0436] Upon stimulation with target cells, B7-H3 CAR-T cells can release large amounts of Th1 cytokines, including interferon (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor (TNF-a), while only secreting small amounts of Th2 cytokines, including interleukins 4, 6, and 10 (IL-4, IL-6, and IL-10). When co-incubated with B7-H3-positive tumor cell lines, the B7-H3 CAR-PDCA7R cell group induced significantly higher levels of various cytokines than the other B7-H3 CAR-T cell groups. This suggests that B7-H3 CAR-T cells are specific for the B7-H3 antigen and that the B7-H3 CAR-PDCA7R has a higher activation function.

[0437] Example 8 CAR-T cells kill tumor cells

[0438] CAR-T cells of various structures were co-incubated with target tumor cells at different ratios and stained with reagents / kits such as DAPI or Annexin V. The killing function of CAR-T cells against target tumor cells was then detected by flow cytometry.

[0439] B7-H3 CAR-T cells with various structures can effectively kill tumor cells expressing B7-H3, and their killing function is antigen-specific.

[0440] Example 9 Anti-tumor effect of CAR-T cells in animal models

[0441] Various subcutaneous tumor models, breast cancer models, peritoneal metastasis models, and lung metastasis models were established by injecting corresponding B7-H3-expressing tumor cells into mice subcutaneously, in the mammary fat pad, intraperitoneally, or through the tail vein. CAR-T cells or CAR-T cells co-expressing these molecules were injected intravenously or intraperitoneally for treatment (alone or in combination). The efficacy of CAR-T cell therapy was analyzed by measuring tumor growth, mouse survival time, cytokine secretion in mouse serum, tumor infiltration, and in vivo proliferation of CAR-T effector cells.

[0442] Intraperitoneal injection of B7-H3 CAR-T cells can effectively inhibit the growth of peritoneal metastatic malignant tumors and ultimately eliminate the tumor burden.

[0443] B7-H3 CAR-T cells effectively inhibited the growth of transplanted subcutaneous tumors, with a significant upregulation of IFN-γ secretion. Furthermore, CAR-T cells co-expressing these molecules, including B7-H3 CAR-PD28 and B7-H3-CAR-PDCA7R T cells, demonstrated enhanced therapeutic efficacy. Third-generation B7-H3 CAR-T cells combined with PD-1 antibodies effectively inhibited tumor growth and ultimately eliminated tumor burden.

[0444] B7-H3 CAR-T cells can effectively inhibit the growth of orthotopic breast xenografts in vivo and maintain a sustained inhibitory effect on tumor growth. B7-H3-CAR-PDCA7R T cells have a better effect on promoting tumor regression than B7-H3 CAR-T cells.

[0445] B7-H3 CAR-T cell therapy effectively inhibited lung metastasis and prolonged mouse survival, while mice in the control group developed significant lung metastasis and had a shorter survival. B7-H3 CAR-PDCA7R T cells were more effective in inhibiting lung metastasis and prolonging survival than B7-H3 CAR-T cells.

[0446] Example 10 Humanized Antibodies and Activity Assays

[0447] The variable region FR sequences of the obtained murine anti-B7-H3 antibodies were compared and screened with human FR sequences. After humanization mutation of the FR region sequences, humanized anti-B7-H3 antibody sequences of three light chains (L1, L2, L3) and three heavy chains (H1, H2, H3) were obtained. Furthermore, the affinity constants of the nine single-chain antibodies formed by the combination of each light chain and heavy chain were detected by Biacore.

[0448] At the same time, the single-chain antibody sequences of each combination are used to construct CAR-T cells, and the killing function of each combination of CAR-T cells on target tumor cells can be detected and compared through killing experiments. Compared with single-chain antibodies with weak affinity (such as L1 / H1 combination), CAR-T cells constructed with single-chain antibodies with high affinity (such as L2 / H2 combination) generally have higher killing efficacy against target cells. Therefore, selecting single-chain antibodies with appropriate affinity (for example, H2 / L2 or H2 / L1) for CAR-T cell construction may not only maintain the function of CAR-T cells to effectively kill target tumor cells, but also reduce side effects such as off-target damage to normal tissues.

[0449] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> Fuzhou Tuoxintiancheng Biotechnology Co., Ltd. <120> Anti-B7-H3 monoclonal antibodies and their applications in cell therapy <130> P2018-0878 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> artificial sequence <400> 1 Gly Leu Ser Phe Thr Gly Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <400> 2 Gly Tyr Thr Phe Arg Asn Tyr Gly 1 5 <210> 3 <211> 8 <212> PRT <213> artificial sequence <400> 3 Gly Tyr Thr Phe Ser Ser Tyr Trp 1 5 <210> 4 <211> 8 <212> PRT <213> artificial sequence <400> 4 Ile Asn Phe Tyr Thr Gly Ala Thr 1 5 <210> 5 <211> 8 <212> PRT <213> artificial sequence <400> 5 Ile Asn Thr Tyr Thr Gly Glu Pro 1 5 <210> 6 <211> 8 <212> PRT <213> artificial sequence <400> 6 Ile Asp Pro Thr Ser Gly Asn Thr 1 5 <210> 7 <211> 9 <212> PRT <213> artificial sequence <400> 7 Ala Ile Leu Gly Phe Ala Leu Asp Tyr 1 5 <210> 8 <211> 11 <212> PRT <213> artificial sequence <400> 8 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr 1 5 10 <210> 9 <211> 12 <212> PRT <213> artificial sequence <400> 9 Ala Arg Ile Phe Met Val Pro Tyr Tyr Phe Ala Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> artificial sequence <400> 10 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 11 <211> 11 <212> PRT <213> artificial sequence <400> 11 Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr 1 5 10 <210> 12 <211> 5 <212> PRT <213> artificial sequence <400> 12 Ser Ser Val Ser Tyr 1 5 <210> 13 <211> 3 <212> PRT <213> artificial sequence <400> 13 Lys Ile Ser 1 <210> 14 <211> 3 <212> PRT <213> artificial sequence <400> 14 Gln Met Ser 1 <210> 15 <211> 3 <212> PRT <213> artificial sequence <400> 15 Leu Thr Ser 1 <210> 16 <211> 9 <212> PRT <213> artificial sequence <400> 16 Ser Gln Gly Thr His Val Pro Leu Thr 1 5 <210> 17 <211> 9 <212> PRT <213> artificial sequence <400> 17 Ala Gln Asn Leu Glu Leu Pro Leu Thr 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <400> 18 Gln Gln Trp Ser Ser Asp Pro Leu Thr 1 5 <210> 19 <211> 119 <212> PRT <213> Artificial sequence <400> 19 Val His Ser Ala Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 1 5 10 15 Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Leu Ser Phe 20 25 30 Thr Gly Tyr Tyr Met His Trp Val Thr Gln Ser Pro Gly Arg Ser Leu 35 40 45 Glu Trp Ile Ala Tyr Ile Asn Phe Tyr Thr Gly Ala Thr Thr Tyr Asn 50 55 60 Gln Lys Phe Met Gly Lys Ala Thr Phe Thr Val Asp Pro Ser Ser Asn 65 70 75 80 Thr Ala Tyr Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 85 90 95 Tyr Tyr Cys Ala Ile Leu Gly Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 20 <211> 118 <212> PRT <213> Artificial sequence <400> 20 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Lys Gln Thr Pro Gly Lys Asp Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Arg Gly Arg Phe Thr Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Lys Tyr Phe Cys 85 90 95 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 21 <211> 119 <212> PRT <213> Artificial sequence <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Thr Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Phe Met Val Pro Tyr Tyr Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Ala Leu Thr Ile Ser Ser 115 <210> 22 <211> 118 <212> PRT <213> artificial sequence <400> 22 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 23 <211> 118 <212> PRT <213> artificial sequence <400> 23 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Gln Asp Phe 50 55 60 Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 118 <212> PRT <213> artificial sequence <400> 24 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Gln Asp Phe 50 55 60 Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr Trp Gly Glnx Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 25 <211> 113 <212> PRT <213> artificial sequence <400> 25 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Note: There seems to be a typo in the original text where "Glnx" is present in line . It is translated as "Gln" in the English version assuming it's a typo.Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Gly 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg <210> 26 <211> 113 <212> PRT <213> artificial sequence <400> 26 Asp Val Val Met Thr Gln Thr Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Ser Lys Leu Glu Leu Lys 100 105 110 Angry <210> 27 <211> 107 <212> PRT <213> artificial sequence <400> 27 Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Asn Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asp Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 28 <211> 113 <212> PRT <213> Artificial sequence <400> 28 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 29 <211> 113 <212> PRT <213> Artificial sequence <400> 29 Asp Val Val Met Thr Gln Ser Pro Leu Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser<000141​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 31 <211> 21 <212> PRT <213> artificial sequence <400> 31 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 20 <210> 32 <211> 135 <212> PRT <213> artificial sequence <400> 32 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55 60 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 115 120 125 Ala Glu Val Pro Thr Ala His 130 135 <210> 33 <211> 59 <212> PRT <213> artificial sequence <400> 33 Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr[[ID=!44]] 1 5 10 15 Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro 20 25 30 Ile Leu Leu Thr Cys Pro Thr Ile Ser Ile Leu Ser Phe Phe Ser Val 35 40 45 Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp 50 55 <210> 34 <211> 195 <212> PRT <213> artificial sequence <400> 34 Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys 1 5 10 15 Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val 20 25 30 Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp 35 40 45 Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe 50 55 60 Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val 65 70 75 80 Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser 85 90 95 Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala 100 105 110 Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu 115 120 125 Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu 130 135 140 Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly 145 150 155 160 Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser 165 170 175 Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr 180 185 190 Gln Asn Gln 195 <210> 35 <211> 404 <212> PRT <213> Artificial sequence <400> 35 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55 60 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 115 120 125 Ala Glu Val Pro Thr Ala His Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Pro Asp His Tyr Phe Lys Gly Phe Trp Ser 145 150 155 160 Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser 165 170 175 Ser Gly Glu Met Asp Pro Ile Leu Leu Thr Cys Pro Thr Ile Ser Ile 180 185 190 Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu 195 200 205 Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His 210 215 220 Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn 225 230 235 240 Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val 245 250 255 Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr 260 265 270 Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp 275 280 285 Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu 290 295 300 Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser 305 310 315 320 Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg 325 330 335 Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser 340 345 350 Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser 355 360 365 Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr 370 375 380 Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe 385 390 395 400 Tyr Gln Asn Gln <210> 36 <211> 1203 <212> DNA <213> artificial sequence <400> 36 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 60 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 120 gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 180 gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 240 cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 300 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 360 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccacggtgg aggcggttca 420 ggcggaggtg gctctggcgg tggcggatcg cctgatcact attttaaagg cttctggagt 480 gaatggagtc caagttatta cttcagaact ccagagatca father aggggagatg gatcctatct tactaaccat cagcattttg agttttttct ctgtcgctct gttggtcatc 600 ttggcctgtg tgttatggaa aaaaaggatt aagcctatcg tatggcccag tctccccgat 660 cataagaaga ctctggaaca tctttgtaag aaaccaagaa aaaatttaaa tgtgagtttc aatcctgaaa gtttcctgga ctgccagatt catagggtgg atgacattca agctagagat 780 840. gttttctgca agatacgttt cctcagcaac tagaagaatc tgagaagcag aggcttggag gggatgtgca gagccccaac tgcccatctg aggctgtagt catcactcca gaaagctttg gaagagattc atccctcaca tgcctggctg ggaatgtcag tgcatgtgac gcccctattc tctcctcttc caggtcccta gactgcaggg agagtggcaa gaatgggcct catgtgtacc aggacctcct gcttagcctt gggactacaa acagcacgct gccccctcca ttttctctcc aatctggaat cctgacattg aacccagttg ctcagggtca gcccattctt 1140 acttccctgg gatcaaatca agaagaagca tatgtcacca tgtccagctt ctaccaaaac 1200 cag 1203 <210> 37 <211> 934 <212> PRT <213> Artificial Sequence <400> 37 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Arg Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Gln Asp Phe 50 55 60 Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Leu Arg His His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Asn 130 135 140 Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys 145 150 155 160 Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln 165 170 175 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu 180 185 190 Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 210 215 220 Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 245 250 255 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 260 265 270 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 275 280 285 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 290 295 300 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 305 310 315 320 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 325 330 335 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 340 345 350 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 385 390 395 400 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 405 410 415 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 420 425 430 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 435 440 445 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 450 455 460 Leu Pro Pro Arg Arg Ala Lys Arg Gly Lys Pro Ile Pro Asn Pro Leu 465 470 475 480 Leu Gly Leu Asp Ser Thr Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu 485 490 495 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu 500 505 510 Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala 515 520 525 Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro 530 535 540 Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala 545 550 555 560 Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn 565 570 575 Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe 580 585 590 Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr 595 600 605 Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg 610 615 620 Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro 625 630 635 640 Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu 645 650 655 Arg Arg Ala Glu Val Pro Thr Ala His Gly Gly Gly Gly Ser Gly Gly 660 665 670 Gly Gly Ser Gly Gly Gly Gly Ser Pro Asp His Tyr Phe Lys Gly Phe 675 680 685 Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr Pro Glu Ile Asn 690 695 700 Asn Ser Ser Gly Glu Met Asp Pro Ile Leu Leu Thr Cys Pro Thr Ile 705 710 715 720 Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys 725 730 735 Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro 740 745 750 Asp His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn 755 760 765 Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His 770 775 780 Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln 785 790 795 800 Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly 805 810 815 Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr 820 825 830 Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn 835 840 845 Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp 850 855 860 Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu 865 870 875 880 Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu 885 890 895 Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile 900 905 910 Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser 915 920 925 Ser Phe Tyr Gln Asn Gln 930 <210> 38 <211> 2793 <212> DNA <213> artificial sequence <400> 38 gatgttgtga tgacgcagag tccactctcc aatccagtca ctcttggaac atcagcttcc 60 atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120 tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgatttcac actgaagatc 240 agcagagtgg aggctgagga tgtgggtgtt tattactgtg ctcaaaatct tgaacttccg 300 ctcacgttcg gtcaggggac caagctggag atcaaaggtg gaggcggttc aggcggaggt 360 ggctctggcg gtggcggatc gcagatccag ttggtgcagt ctggatctga actgaagaag 420 cctggagcgt cagtcaaggt ctcctgcaag gttctgggt ataccttcag aaactatgga 480 atgagctggg tgaggcaggc tccaggacag ggtttagagt ggatgggctg gataaacacc 540 tacactggag agccaacata tgctcaagac ttcaggggac ggtttgtctt ctctttggat 600 acctctgtca gcactgccta tttgcagatc agtagcctca aagctgagga cacggctgtc 660 tattactgtg caagatggtt acgacaccat gctatggact actggggtca aggaaccttg 720 gtcaccgtct cctcaaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 780 gcgtcgcagc ccctgtccct gcgcccagag gcgtcccggc cagcggcggg gggcgcagtg 840 cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 900 tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 960 ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1020 ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgcg ggtgaagttc 1080 agccggagcg ccgacgcccc tgcctaccag cagggccaga accagctgta caacgagctg 1140 aacctgggcc ggaggagga gtacgacgtg ctggacaagc ggagaggccg ggaccctgag atgggcggca agccccggag aaagaaccct caggagggcc tgtataacga actgcagaaa gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gaggggcaag 1380. ggccacgacg gcctgtacca gggcctgagc accgccacca aggataccta cgacgccctg cacatgcagg ccctgccccc cagaagagcc aagcggggta agcctatccc taaccctctc ctcggtctcg attctcgag cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaccc tggacctatg gccctgcccg tgaccgccct gctgctgccc 1560 ctggccctgc tgctgcacgc cgccaggccg ccaggatggt tcttagactc cccagacagg 1620 ccctggacc cccccacctt ctccccagcc ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca gagctcaggg tgacagagag aagggcagaa gtgcccacag cccacggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcg cctgatcact attttaaagg cttctggagt gaatggagtc caagttatta cttcagaact ccagagatca father aggggagatc gatcctatct tactaaccat cagcattttg agttttttct ctgtcgctct gttggtcatc ttggcctgtg tgttatggaa aaaaaggatt 2220 aagcctatcg tatggcccag tctccccgat cataagaaga ctctggaaca tctttgtaag 2340. aaaccaaga aaaatttaa tgtgagtttc aatcctgaa gtttcctgga ctgccagatt catagggtgg atgacattca agctagagat gaagtggaag gttttctgca agatacgttt cctcagcaac tagaagaatc tgagaagcag aggcttggag gggatgtgca gagccccaac tgcccatctg aggatgtagt catcactcca gaaagctttg gaagagattc atccctcaca tgcctggctg ggaatgtcag tgcatgtgac gcccctattc tctcctcttc caggtcccta 2580 gactgcaggg agagtggcaa gaatgggcct catgtgtacc aggacctcct gcttagcctt 2640 gggactacaa acagcacgct gccccctcca ttttctctcc aatctggaat cctgacattg 2700 aacccagttg ctcagggtca gcccattctt acttccctgg gatcaaatca agaagaagca 2760 tatgtcacca tgtccagctt ctaccaaaac cag 2793 <210> 39 <211> 546 <212> DNA <213> Artificial Sequence <400> 39 aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360 tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540 gcctac 546

Claims

1. An anti-B7-H3 antibody, characterized in that The antibody has: (1) A heavy chain variable region, comprising the following three complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:7; (2) A light chain variable region, wherein the light chain variable region includes the following three complementarity determining regions (CDRs): CDR1' shown in SEQ ID NO: 10, CDR2' shown in SEQ ID NO: 13, and CDR3' shown in SEQ ID NO:

16.

2. The antibody according to claim 1, wherein The antibody has a heavy chain having the heavy chain variable region and a heavy chain constant region; and a light chain having the light chain variable region and a light chain constant region.

3. The antibody according to claim 1, wherein The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

19.

4. The antibody according to claim 1, wherein The light chain variable region has the amino acid sequence shown in SEQ ID NO:

25.

5. A recombinant protein, characterized in that The recombinant protein has: (i) the antibody of claim 1; and (ii) an optional tag sequence to facilitate expression and / or purification.

6. A CAR construct, characterized in that The antigen binding region of the CAR construct is a scFv that specifically binds to B7-H3, and the scFv has a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:7; The light chain variable region includes the following three complementarity determining regions (CDRs): CDR1' shown in SEQ ID NO: 10, CDR2' shown in SEQ ID NO: 13, and CDR3' shown in SEQ ID NO:

16.

7. An engineered immune cell, characterized in that The immune cells include: (a) a first expression cassette for expressing an exogenous CAR construct according to claim 6; and (b) optionally, a second expression cassette that expresses a fusion protein comprising PD1-CD28 or PD1-IL7R.

8. An antibody-drug conjugate, characterized in that: The antibody drug conjugate contains: (a) an antibody portion selected from the group consisting of: The antibody according to claim 1; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

9. Use of the antibody according to claim 1, the recombinant protein according to claim 5, the CAR construct according to claim 6, the immune cell according to claim 7, or the antibody-drug conjugate according to claim 8, characterized in that: For (i) preparing a drug or preparation for preventing and / or treating cancer or tumors; and / or (ii) preparing a detection reagent or kit; The cancer or tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof; The blood tumor is selected from the group consisting of acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or a combination thereof; The solid tumor is selected from the following group: gastric cancer, liver cancer, kidney tumor, lung cancer, bone cancer, colorectal cancer, breast cancer, cervical cancer, ovarian cancer, lymphoma, bladder tumor, nervous system tumor, solid tumor abdominal metastasis, or a combination thereof; wherein the bone cancer includes osteosarcoma; the nervous system tumor includes glioma and neuroblastoma.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition include: The antibody of claim 1, the recombinant protein of claim 5, the CAR construct of claim 6, the immune cell of claim 7, and / or the antibody-drug conjugate of claim 8, and a pharmaceutically acceptable carrier.

11. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) The antibody of claim 1; (2) the recombinant protein according to claim 5; and (3) The CAR construct of claim 6.

12. A carrier, characterized in that The vector contains the polynucleotide according to claim 11.

13. A genetically engineered host cell, characterized in that The host cell contains the vector according to claim 12 or the polynucleotide according to claim 11 is integrated into its genome.

14. A method for preparing engineered immune cells, characterized in that: The following steps are involved: (A) Providing an immune cell to be modified; and (B) introducing a first expression cassette and an optional second expression cassette into the immune cell to be modified, wherein the first expression cassette expresses the CAR construct according to claim 6, and the second expression cassette expresses a fusion protein comprising PD1-CD28 or PD1-IL7R, thereby obtaining an engineered immune cell.

15. A non-diagnostic in vitro method for detecting B7-H3 protein in a sample, characterized in that: The method comprises the steps of: (1) contacting the sample with the antibody according to claim 1 in vitro; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of B7-H3 protein in the sample.

16. A detection board, characterized in that: The detection plate comprises: a substrate and a test strip, wherein the test strip contains the antibody according to claim 1 or the antibody-drug conjugate according to claim 8.

17. A kit for preparing the engineered immune cells according to claim 7, characterized in that: The kit comprises: (a) a first container, and a first nucleic acid sequence located in the first container, wherein the first nucleic acid sequence comprises a first expression cassette for expressing the CAR construct of claim 6; and Optionally, (b) a second container, and a second nucleic acid sequence located in the second container, wherein the second nucleic acid sequence comprises a second expression cassette for expressing the fusion protein.

18. A diagnostic kit, characterized in that include: (1) a first container containing the antibody of claim 1; and (2) A second container containing a secondary antibody against the antibody of claim 1.

Images