CN114773477B - anti-B7-H3 antibodies and uses thereof - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to an anti-B7-H3 antibody and application thereof, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, the anti-B7-H3 antibody heavy chain variable region comprises a CDR-H1 with an amino acid sequence shown as SEQ ID No.5, a CDR-H2 with an amino acid sequence shown as SEQ ID No.6 and a CDR-H3 with an amino acid sequence shown as SEQ ID No.7, and the light chain variable region comprises a CDR-L1 with an amino acid sequence shown as SEQ ID No.8, a CDR-L2 with an amino acid sequence shown as SEQ ID No.9 and a CDR-L3 with an amino acid sequence shown as SEQ ID No. 10. The anti-B7-H3 antibody can be used for preparing or screening tumor treatment or tumor diagnosis medicines.

Description

anti-B7-H3 antibodies and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an anti-B7-H3 antibody and application thereof.

Background

B7-H3 is a type I transmembrane protein, belonging to the family of B7 immune co-stimulatory and co-inhibitory molecules. There are two subtypes, 2Ig-B7-H3 and 4Ig-B7-H3, in humans and only 2Ig-B7-H3 in mice. In non-malignant tissues, B7-H3 mainly plays a role in immunosuppression, inhibits activation and proliferation of T cells, and inhibits cytotoxic activity of natural killer cells. The B7-H3 protein is highly expressed in many different types of human malignancies. In malignant tissues, B7-H3 inhibits tumor antigen-specific immune response, promotes migration and invasion of tumor cells, angiogenesis, chemotherapy resistance and transformation of endothelial cells to stroma, and influences the metabolism of tumor cells. In addition, tumor-associated vessels also overexpress B7-H3. Overexpression of B7-H3 in tumor cells has been associated with a decrease in tumor infiltrating lymphocytes, an accelerated progression of cancer progression, and adverse clinical consequences for some malignancies, such as pancreatic ductal adenocarcinoma, prostate cancer, ovarian cancer, lung cancer, and clear cell renal carcinoma. Therefore, B7-H3 is a very potential tumor immunotherapy target, and the development of B7-H3-targeted therapies is in line with the current need.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide an anti-B7-H3 antibody and its use for solving the problems in the prior art.

To achieve the above and other related objects, the present invention provides an anti-B7-H3 antibody, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, and the anti-B7-H3 antibody has one or more of the following technical characteristics;

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence shown in SEQ ID No. 5;

<2> the variable region of the heavy chain comprises CDR-H2 having an amino acid sequence shown in SEQ ID No. 6;

<3> the heavy chain variable region comprises CDR-H3 having an amino acid sequence shown in SEQ ID No. 7;

<4> the variable region of the light chain comprises CDR-L1 having an amino acid sequence shown in SEQ ID No. 8;

<5> the variable region of the light chain comprises CDR-L2 having an amino acid sequence shown in SEQ ID No. 9;

<6> the variable region of the light chain comprises CDR-L3 having an amino acid sequence shown in SEQ ID No. 10.

Preferably, the anti-B7-H3 antibody is a single chain antibody.

Preferably, the amino acid sequence of the anti-B7-H3 single-chain antibody is shown in SEQ ID No. 23.

The invention also provides application of the anti-B7-H3 antibody in preparing or screening tumor treatment medicines or preparing tumor diagnosis medicines.

The invention also provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the anti-B7-H3 antibody.

The invention also provides an isolated polynucleotide encoding the heavy chain variable region and/or the light chain variable region of the anti-B7-H3 antibody, or encoding the isolated polypeptide.

The invention also provides a nucleic acid construct comprising the isolated polynucleotide.

The invention also provides a T lymphocyte which expresses the polypeptide.

As described above, the anti-B7-H3 antibody and the use thereof of the present invention have the following beneficial effects: better cell killing effect.

Drawings

FIG. 1 provides readings from anti-human B7-H3 Fc monoclonal phage ELISA 96-well plates.

Figure 2 provides a schematic diagram of the pCA70-CAR vector structure for B7-H3 CAR mRNA generation.

FIG. 3 provides B7-H3 mRNA expression levels of various tumor cells relative to Jeko1 cells.

FIG. 4 provides the expression levels of B7-H3 proteins on the surface of various tumor cells.

FIG. 5 provides binding of T cells to different concentrations of human 2Ig-B7-H3-Fc protein following electroporation of B7-H3 CAR mRNA.

Figure 6 provides the proportion of T cells expressing B7-H3 CAR after electroporation of B7-H3 CAR mRNA.

Figure 7 provides killing curves of T cells against various B7-H3 positive tumor cells following B7-H3 CAR mRNA electroporation.

Figure 8 provides the proportion of T cells expressing B7-H3 CAR after electroporation of B7-H3 CAR mRNA.

Figure 9 provides killing curves of T cells against various tumor cells following B7-H3 CAR mRNA electroporation.

FIG. 10 provides the levels of B7-H3 expression by varying amounts of B7-H3 mRNA electroporated Jeko1 cells.

Figure 11 provides the proportion of T cells expressing B7-H3 CAR after electroporation of B7-H3 CAR mRNA.

Figure 12 provides the ratio of CD107a expression after co-culture of B7-H3 CAR mRNA electroporated T cells with varying amounts of B7-H3 mRNA electroporated Jeko1 cells.

Figure 13 provides killing curves of T cells after electroporation of B7-H3 CAR mRNA against tumor cells with different levels of antigen expression.

Detailed Description

The terms "anti-B7-H3 antibody" and "anti-B7-H3 antibody" are used interchangeably herein.

The term "antibody" is used herein in its broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An antibody that "binds" an antigen of interest, such as the B7-H3 antigen, refers to an antibody that is capable of binding the antigen with sufficient affinity such that the antibody can be used as a therapeutic agent to target cells expressing the antigen. If the antibody is one that binds B7-H3, it typically binds preferentially to B7-H3, but not to other antibodies.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, typically in minor amounts, except for possible naturally occurring mutations. Monoclonal antibodies are highly specific, i.e., directed against a single antigenic site. In addition, unlike polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies offer the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Monoclonal antibodies herein expressly include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

An "intact" antibody is one that comprises an antigen-binding variable region as well as a light chain constant region (C) and heavy chain constant regions CH1, CH2, andCH 3. The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, Fv fragments, linear antibodies, single chain antibodies (scFv).

An "Fv" fragment is an antibody fragment that comprises the entire antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable region in close association, which may be covalent (as in scFv). In this configuration, the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer.

The "Fab" fragment includes the variable and constant regions of the light chain and the variable and first constant regions of the heavy chain (CH 1). F (ab') 2 antibody fragments comprise a pair of Fab fragments, which are typically covalently linked near their carboxy termini by a hinge cysteine between them. Other chemical coupling methods for antibody fragments are also known in the art.

A "single chain antibody" or fragment of an antibody referred to as an "scFv" comprises the VH and VL domains of an antibody. Typically, single chain antibodies also comprise a polypeptide linker (linker) between the VH and VL domains that allows the scFv to form the desired structure for binding to the antigen.

The term "linear antibody" comprises pairs of tandem Fd segments (VH-CH 1-VH-CH 1) that form pairs of antigen-binding regions with complementary light chain polypeptides. Linear antibodies can be bispecific or monospecific.

The term "antibody variable region" as used herein refers to the portions of the light and heavy chains of an antibody molecule that include the amino acid sequences of the complementarity determining regions (CDRs: i.e., CDR1, CDR2 and CDR 3) and Framework Regions (FRs). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. According to the method used in the present invention, the amino acid positions designated by the CDRs and FRs may be defined by Kabat et al (numbering system described in Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service National Institutes of Health, Bethesda, Md. (1991)).

The term "complementarity determining regions" (CDRs: i.e., CDR1, CDR2 and CDR 3) as used herein refers to the amino acid residues of the variable region of an antibody, the presence of which is essential for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, andCDR 3. Each CDRs may comprise amino acid residues from the "CDRs" as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region).

The "framework regions" (hereinafter FR) are those variable region residues other than the CDR residues. Each variable region typically has four FR of FR1, FR2, FR3 andFR 4. If the CDRs are defined by Kabat, the light chain FR residues are located at approximately residues 1-23 (LCFR 1), 35-49 (LCFR 2), 57-88 (LCFR 3), and 98-107 (LCFR 4), and the heavy chain FR residues are located at approximately residues 1-30 (HCFR 1), 36-49 (HCFR 2), 66-94 (HCFR 3), and 103-113 (HCFR 4) of the heavy chain residues. If the CDRs contain amino acid residues from hypervariable loops, the light chain FR residues are located substantially at residues 1-25 (LCFR 1), 33-49 (LCFR 2), 53-90 (LCFR 3), and 97-107 (LCFR 4) in the light chain, and the heavy chain FR residues are located substantially at residues 1-25 (HCFR 1), 33-52 (HCFR 2), 56-95 (HCFR 3), and 102-113 (HCFR 4) in the heavy chain. In some cases, when the CDRs comprise those amino acids from the CDRs and hypervariable loops defined according to Kabat, the FR residues will be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, heavy chain FR1 residues are at positions 1-25 and FR2 residues are at positions 36-49.

"T cell epitope" (T cell epitope) as used herein refers to a possible peptide fragment of a monoclonal antibody that is bound and presented by MHC molecules as a protein antigen itself and recognized by a T cell antigen receptor. These peptides contained in monoclonal therapeutic antibodies increase the immune response of the patient to the therapeutic antibody. The greater the number of these peptides, the higher the probability of eliciting an immune response.

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that contain, at a minimum, sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Typically, the humanized antibody will comprise substantially no less than at least one, and typically two, variable regions in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. Optionally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

The invention provides an anti-B7-H3 antibody, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, and the anti-B7-H3 antibody has one or more of the following technical characteristics;

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence shown in SEQ ID No. 5;

<2> the variable region of the heavy chain comprises CDR-H2 having an amino acid sequence shown in SEQ ID No. 6;

<3> the heavy chain variable region comprises CDR-H3 having an amino acid sequence shown in SEQ ID No. 7;

<4> the variable region of the light chain comprises CDR-L1 having an amino acid sequence shown in SEQ ID No. 8;

<5> the variable region of the light chain comprises CDR-L2 having an amino acid sequence shown in SEQ ID No. 9;

<6> the variable region of the light chain includes CDR-L3 having an amino acid sequence shown in SEQ ID No. 10.

In certain embodiments of the invention, the complementarity determining regions of the heavy chain variable region of the anti-B7-H3 antibody include CDR-H1 having an amino acid sequence shown in SEQ ID No.5, CDR-H2 having an amino acid sequence shown in SEQ ID No.6, and CDR-H3 having an amino acid sequence shown in SEQ ID No. 7.

In certain embodiments of the invention, the complementarity determining regions of the light chain variable region of the anti-B7-H3 antibody include CDR-L1 having an amino acid sequence shown in SEQ ID No.8, CDR-L2 having an amino acid sequence shown in SEQ ID No.9, and CDR-L3 having an amino acid sequence shown in SEQ ID No. 10.

In certain embodiments of the present invention, the complementarity determining regions of the heavy chain variable region comprise CDR-H1 having an amino acid sequence shown in SEQ ID No.5, CDR-H2 having an amino acid sequence shown in SEQ ID No.6 and CDR-H3 having an amino acid sequence shown in SEQ ID No.7, and the complementarity determining regions of the light chain variable region comprise CDR-L1 having an amino acid sequence shown in SEQ ID No.8, CDR-L2 having an amino acid sequence shown in SEQ ID No.9 and CDR-L3 having an amino acid sequence shown in SEQ ID No. 10.

In certain embodiments of the invention, framework regions are also included in the heavy chain variable region and the light chain variable region. In some embodiments of the invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3) amino acids to the sequence of the framework region of the murine monoclonal antibody variable region, and the sequence of the framework region may have a homology of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with the sequence of the framework region of the human monoclonal antibody variable region.

In certain embodiments of the invention, the amino acid sequence of HCFR1 is set forth as SEQ ID No.11, the amino acid sequence of HCFR2 is set forth as SEQ ID No.12, the amino acid sequence of HCFR3 is set forth as SEQ ID No.13, and the amino acid sequence of HCFR4 is set forth as SEQ ID No. 14. The amino acid sequence of LCFR1 is shown as SEQ ID No.15, the amino acid sequence of LCFR2 is shown as SEQ ID No.16, the amino acid sequence of LCFR3 is shown as SEQ ID No.17, and the amino acid sequence of LCFR4 is shown as SEQ ID No. 18.

In certain embodiments of the invention, the anti-B7-H3 antibody is a single chain antibody (scFv). The anti-B7-H3 single-chain antibody comprises V_H And V_L ，V_H And V_L A connecting peptide is arranged between the two, and the anti-B7-H3 single-chain antibody can sequentially comprise V from the N end to the C end_L Linker peptide and V_H The anti-B7-H3 single-chain antibody also can sequentially comprise V from N end to C end_H Linker peptide and V_L . The linker peptide can be any of a variety of linker peptides suitable for use in the art for forming scfvs, for example, the linker peptide can be G4S3 linker, which G4S3 linker can be selected or designed as described in Michel Sadelain et al, Science relative Medicine, 2013; carl H, June etc., Science relative Medicine, 2015.

In certain embodiments of the invention, the anti-B7-H3 single chain antibody is selected from a human phage antibody display library. The amino acid sequences of the light chain variable region and the heavy chain variable region of the anti-B7-H3 single-chain antibody are shown as SEQ ID No.19 and SEQ ID No. 20. The heavy chain variable region and light chain variable region nucleotide sequences of the anti-B7-H3 single-chain antibody are respectively shown as SEQ ID No.21 and SEQ ID No. 22.

In certain embodiments of the invention, the amino acid sequence of the anti-B7-H3 single chain antibody is set forth in SEQ ID No. 23.

The invention also provides application of the anti-B7-H3 antibody in preparation or screening of therapeutic drugs or preparation of diagnostic drugs.

The therapeutic drug can be a drug which takes the B7-H3 antigen as an action target, binds or acts on the B7-H3 antigen, and thus treats and/or prevents indications.

In certain embodiments of the invention, the therapeutic agent may be a tumor therapeutic agent. The tumor treatment drug can be a drug which takes the B7-H3 antigen on the functional surface of the surface of a tumor cell as a target, and binds or acts on the B7-H3 antigen so as to treat and/or prevent tumors. The tumor can be a solid tumor or a blood tumor positive for B7-H3 expression. The solid tumor is lung cancer, ovarian cancer, breast cancer and pancreatic cancer. Such hematological tumors include, but are not limited to, non-hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, human B cell precursor leukemia, multiple myeloma, malignant lymphoma, and the like.

In certain embodiments of the invention, the therapeutic agent is a chimeric antigen receptor cell therapeutic agent.

The chimeric antigen receptor cell therapeutic agents typically include chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and the like. The chimeric antigen receptor T cells generally include T lymphocytes and chimeric antigen receptors. The chimeric antigen receptor NK cells generally include NK cells and a chimeric antigen receptor. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain. In certain embodiments of the invention, the extracellular domain comprises the anti-B7-H3 antibody, i.e., the chimeric antigen receptor cell can express the anti-B7-H3 antibody on the cell surface, which can direct the cell to act on cells expressing the B7-H3 antigen (e.g., tumor cells). The action on the cell expressing the B7-H3 antigen may be killing the cell expressing the B7-H3 antigen, or the like.

The diagnostic drug is a reagent for diagnosing an action target B7-H3 antigen and B7-H3 antigen serving as a biomarker.

The invention also provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the anti-B7-H3 antibody.

In certain embodiments of the invention, the polypeptide is a chimeric antigen receptor.

In certain embodiments of the invention, the transmembrane domain may comprise the transmembrane domain of a protein molecule such as CD8 α, CD28, DAP10, and the like.

For example, the amino acid sequence of CD8 α is shown in SEQ ID No. 31. The nucleotide sequence of CD8 alpha is shown in SEQ ID No. 26. For another example, the sequence of CD8 α can be referenced to NM _001145873, the sequence of CD28 can be referenced to NM _006139, and the sequence of DAP10 can be referenced to NM _ 014266.

In certain embodiments of the invention, the endodomain may include a costimulatory domain and/or a signaling domain, e.g., the endodomain may include the signaling domain of a 4-1BB, CD28, OX40, ICOS, CD3 zeta, DAP10, etc. protein molecule. For example, the amino acid sequence of 4-1BB is shown in SEQ ID No. 32. The nucleotide sequence of 4-1BB is shown as SEQ ID No. 27. The amino acid sequence of CD3 zeta is shown in SEQ ID No. 33. The nucleotide sequence of CD3 zeta is shown in SEQ ID No. 28. For another example, the sequence of 4-1BB may refer to NM _001561, the sequence of CD28 may refer to NM _006139, the sequence of OX40 may refer to NM _003327, the sequence of ICOS may refer to NM _012092, the sequence of CD3 zeta may refer to NM _198053, and the sequence of DAP10 may refer to NM _ 014266. In one embodiment of the present invention, the endodomain comprises 4-1BB and CD3 zeta in sequence from N-terminus to C-terminus.

In certain embodiments of the invention, the extracellular domain further comprises a hinge region selected from the group consisting ofCD 8. The amino acid sequence of the CD8 hinge is shown in SEQ ID NO. 30. The nucleotide sequence of the CD8 hinge is shown in SEQ ID NO. 25.

In certain embodiments of the invention, the extracellular domain further comprises a Signal Peptide (SP) selected from the group consisting of CD8 SP. The amino acid sequence of CD8 SP is shown in SEQ ID NO. 29. The nucleotide sequence of CD8 SP is shown in SEQ ID NO. 24.

In some embodiments of the invention, the polypeptide comprises the anti-B7-H3 single-chain antibody, a transmembrane domain and an intracellular domain from N end to C end. In some embodiments of the invention, the polypeptide comprises, in order from N-terminus to C-terminus, an anti-B7-H3 single chain antibody, a CD8 a transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In a specific embodiment of the invention, the polypeptide comprises the anti-B7-H3 single-chain antibody, a CD28 transmembrane region, a CD28 costimulatory domain and a CD3 zeta signal domain from N end to C end in sequence. In another embodiment of the invention, the polypeptide comprises the anti-B7-H3 single chain antibody, CD8 a transmembrane region, OX40 costimulatory domain, CD3 zeta signaling domain in order from N-terminus to C-terminus. In another embodiment of the present invention, the polypeptide comprises the anti-B7-H3 single-chain antibody, CD8 a transmembrane region, ICOS costimulatory domain, and CD3 zeta signaling domain in sequence from N-terminal to C-terminal. In another embodiment of the invention, the polypeptide comprises the anti-B7-H3 single-chain antibody, CD8 alpha transmembrane region, 4-1BB costimulatory domain and CD3 zeta from N end to C end. In another embodiment of the present invention, the polypeptide comprises the anti-B7-H3 single chain antibody, CD28 transmembrane region, CD28 costimulatory domain, OX40 costimulatory domain, CD3 zeta signaling domain in sequence from N-terminus to C-terminus.

The invention also provides an isolated polynucleotide encoding the heavy chain variable region and/or the light chain variable region of the anti-B7-H3 antibody, or encoding the isolated polypeptide.

The polynucleotide is DNA or RNA.

The invention also provides a nucleic acid construct comprising the isolated polynucleotide.

The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. The nucleic acid construct is a viral vector or a non-viral vector. Both viral and non-viral vectors include empty vectors and expression frameworks. The viral vector may be a lentiviral vector or an adeno-associated viral vector. The non-viral vector may be an mRNA vector.

The invention also provides a lentivirus which is formed by virus packaging of a lentivirus vector system. The lentivirus can be used to infect T cells to produce CART cells, or to infect NK cells to produce CAR-NK cells.

The invention also provides a cell expressing the polypeptide.

In certain embodiments of the invention, the cell is a CAR T cell.

In certain embodiments of the invention, the polypeptide is a chimeric antigen receptor.

The CART cell can typically express the polypeptide, which can typically bind to B7-H3 antigen, more particularly B7-H3 antigen by comprising the extracellular domain of the anti-B7-H3 antibody, which can typically be activated and/or stimulated to proliferate when the polypeptide binds to the B7-H3 antigen. In certain embodiments of the invention, the extracellular domain comprises the anti-B7-H3 antibody, i.e., the CAR T cells can express the anti-B7-H3 antibody on the surface of T lymphocytes, such that the T lymphocytes can be directed to act on cells expressing the B7-H3 antigen (e.g., tumor cells), which can be killing cells expressing the B7-H3 antigen, etc.

In certain embodiments of the invention, the cell is a CAR NK cell.

In certain embodiments of the invention, the polypeptide is a chimeric antigen receptor.

The CAR NK cells can typically express the polypeptide and can typically bind to B7-H3 antigen, more particularly can bind to B7-H3 antigen via an extracellular domain comprising the anti-B7-H3 antibody, and when the polypeptide binds to the antigen, the CAR NK cells can typically be activated and/or stimulated to proliferate. In certain embodiments of the invention, the extracellular domain comprises the anti-B7-H3 antibody, i.e., the CAR NK cells can express the anti-B7-H3 antibody on the NK cell surface, such that the NK cell can be directed to act on cells expressing the B7-H3 antigen (e.g., tumor cells), which can be killing cells expressing the B7-H3 antigen, etc.

The invention also provides a method for producing the cell, the method comprising transferring the nucleic acid construct to a host cell.

In certain embodiments of the invention, the method of transfer may be electroporation or viral transduction.

In certain embodiments of the invention, the host cell is a T cell or NK cell.

The T cell is a cytotoxic T cell, a helper T cell, a gamma delta cell, a CD4 +/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a T naive cell, an effector T cell, a helper T cell, a memory T cell, a regulatory T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3(Treg) cell, a Th9 cell, a Th17 cell, a Th alpha beta helper cell, a Tfh cell, a stem cell memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.

The invention also provides application of the separated polypeptide, the T lymphocyte and the NK cell in preparing or screening therapeutic drugs or preparing diagnostic drugs.

The therapeutic or diagnostic agent may be one that targets the B7-H3 antigen, binds to or acts on the B7-H3 antigen, and thereby treats and/or prevents the indication.

In certain embodiments of the invention, the therapeutic agent may be a tumor therapeutic agent. The tumor treatment drug can be a drug which takes B7-H3 antigen functionally expressed on the surface of tumor cells as a target, and binds or acts on B7-H3 antigen so as to treat and/or prevent tumors. The tumor can be breast cancer, ovarian cancer, lung cancer and other B7-H3 expression positive tumors.

The invention also provides a diagnostic kit comprising a diagnostically effective dose of the anti-B7-H3 antibody or immunoconjugate thereof. An effective amount generally refers to an amount that provides a diagnostic benefit.

The diagnostic kit can generally aim at the target B7-H3 antigen, and the B7-H3 antigen is used as a biomarker for diagnosis. The diagnostic kit may further comprise a label for the anti-B7-H3 antibody, the label for the anti-B7-H3 antibody can be commonly used for labeling the anti-B7-H3 antibody, and the types of the selectable labels include but are not limited to one or more combinations of fluorescent labels, radioactive labels, enzyme labels, chemiluminescent labels and the like. Depending on the detection principle of the kit, the kit may also typically comprise one or more reagents required for the detection. In addition, the kit can also comprise the following components according to needs: containers, controls (negative or positive controls), buffers, adjuvants, etc., which can be selected by one skilled in the art as appropriate.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Example 1 preparation of human anti-B7-H3 antibody

The following steps were taken to prepare fully human anti-B7-H3 antibody:

1) expression and purification of phage display libraries: log phase TG1 library cultures were infected with freshly thawed M13K07 helper phage with a multiple infection rate of 20: 1 (phage to cell ratio) and induced overnight with IPTG; the phage library was purified by PEG/NaCl precipitation and then the phage titer was determined.

2) Selection of B7-H3-specific scFv-phages: in the first round of selection, Maxisorp plates were coated with 20. mu.g/ml of B7-H3-6His protein overnight at 4 ℃, washed with PBS, blocked with 5% milk +1% BSA in 1 XPBS, incubated in phage solution for 2 hours, and washed 10 times with PBST. The scFv phage bound to the antigen were eluted using acid elution buffer (ph2.2), neutralized, inoculated in 15 ml log phase TG1 culture (OD600=0.5), incubated at 37 ℃ for 30 minutes with shaking for 30 minutes, inoculated onto 2xYT-GA agar plates, and incubated overnight at 30 ℃ for subsequent selection. In subsequent rounds of selection, more stringent selection conditions were used, including decreasing the coated protein concentration (2. mu.g/ml in the second round and 0.5. mu.g/ml in the third round) and increasing the wash cycle (20 in the second round and 30 in the third round).

3) mpELISA screening: after three rounds of selection, 288 positive colonies were selected for monoclonal phage elisa (mpaisa) screening. Phage supernatants generated from individual colony clones were incubated with pre-blocked Maxisorp plates coated with 2. mu.g/mL B7-H3-6His protein. After three washes, 100. mu.l/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in blocking buffer (5% milk +1% BSA, 1 XPBS) was added, followed by incubation at room temperature for 60 min. After washing theplates 5 times with PBST, 100 μ l/well of TMB substrate was addedThe solution was incubated for 10-30 min until a blue color appeared. By adding 50. mu.l/well of stop solution (2N H)₂ SO₄ ) The reaction was terminated. The absorbance at 450nm was read in a microplate reader. Figure 1 shows the absorbance readings from the mpELISA screening. As shown, some positive colonies (absorbance. gtoreq.0.5 at 450 nm) were identified as producing anti-B7-H3 antibody capable of binding to B7-H3-6His protein (FIG. 1).

4) Cloning and sequence analysis: positive clones were screened based on ELISA results and used as templates for PCR cloning of scFv sequences (forward primer sequence shown in SEQ ID NO: 1: tgcagctggcacgacaggtttc, reverse primer sequence shown in SEQ ID NO: 2: cgtcagactgtagcacgtt). Then, the PCR product was sequenced by a sanger sequencing method (forward primer sequence shown as SEQ ID NO: 3: aacaattgaattcaggagga, reverse primer sequence shown as SEQ ID NO: 4: cctcctaagaagcgtagtc). Analyzing CDR regions of the scFv by an abysis website (http:// abysis. org /), wherein the amino acid sequences of the CDRs are shown as SEQ ID NO: 5-10, the amino acid sequences of the framework regions are shown as SEQ ID NO: 11-18, the amino acid sequences of the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID NO: 19-20, and the nucleotide sequences of the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22. The amino acid sequence of scFv is shown in SEQ ID NO: 23.

Example 2 preparation of B7-H3 CAR

Vectors were constructed for the production of anti-B7-H3 CAR mRNA. Firstly, a CD8 signal peptide (namely CD8 SP) with a nucleotide sequence shown as SEQ ID NO:24, a CAR fragment (comprising a CD8 hinge with a nucleotide sequence shown as SEQ ID NO:25, a CD8 transmembrane domain with a nucleotide sequence shown as SEQ ID NO:26, a 4-1BB co-stimulation domain with a nucleotide sequence shown as SEQ ID NO:27, a CD3 zeta signal domain with a nucleotide sequence shown as SEQ ID NO: 28) and a scFv sequence, a CD8 signal peptide, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulation domain and an amino acid sequence of a CD3 zeta signal domain are shown as SEQ ID NO: 29-33, a nucleotide sequence of the scFv is shown as SEQ ID NO:34 are amplified by PCR and cloned to a CAR scFv position of a pCA70 vector through homologous recombination. Figure 2 provides a schematic of the pCA70-CAR vector used to generate CAR mRNA, the nucleotide sequence of the pCA70-CAR vector is shown in SEQ ID No. 35. Second, B7-H3 CAR mRNA was prepared by In Vitro Transcription (IVT). The pCA70-CAR plasmid was linearized by digestion with BspQ1 and checked by running an agarose DNA gel and IVT was performed according to the manufacturer's instructions (Thermofisiher, Cat No: AM 13455). The concentration of the RNA product was determined by nanodrop and checked by running a PAGE gel.

Example 3 detection of expression levels of B7-H3 in tumor cells

1) B7-H3 mRNA transcript levels were measured by RT-qPCR for different tumor cells: diluting cDNA of various tumor cells by 10 times, taking 1 mul of diluted cDNA as a template to prepare 10 mul/well RT-qPCR reaction solution in a 348-well plate, detecting B7-H3 gene by using primers (the sequence is shown as SEQ ID NO: 36-37), detecting beta-actin gene (as an internal reference) by 1 pair of primers (the sequence is shown as SEQ ID NO: 38-39), repeating three wells for each pair of primers, and operating an RT-qPCR program: 95 ℃,2 minutes → (95 ℃, 10 seconds → 60 ℃, 30 seconds) 40 cycles → Curve. The expression of all tumor cell lines B7-H3 mRNA relative to Jeko1 cells was calculated. As shown in FIG. 3, Jeko1, Molm14, Nalm6 and Raji cells did not express B7-H3 mRNA, RPMI8226 expressed B7-H3 mRNA at a very low level, and other cell lines expressed B7-H3 mRNA at a high level.

2) B7-H3 protein levels were measured on the surface of different tumor cells by FACS staining: collecting various tumor cells, incubating and staining with APC-isotype antibody and APC-anti-human B7-H3 antibody, and detecting the staining result by a flow cytometer. As shown in FIG. 4, B7-H3 protein could not be detected on the cell surface of Jeko1, Molm14, Nalm6, RPMI8226 and Raji, other cell lines all expressed B7-H3 protein at high level, and Isotype control was an Isotype antibody.

Example 4 preparation and characterization of B7-H3 CARTs

B7-H3 CAR mRNA was introduced into T cells using the electrotransfer technique by: t cells were collected, washed with Opti-MEM medium, and cultured in Opti-MEM medium at5X 10⁷ Resuspending in/ml; 5 mug of RNA and 100 mug of l T cells were aliquoted, mixed well and electroporated, with the following parameters (BTX electrotransfer apparatus): 500V, 0.7 ms; the cells are then transferred to a pre-warmed chamberOn the medium, the culture was carried out at 37 ℃.

Binding of B7-H3 CAR T cells to 2Ig-B7-H3-Fc recombinant protein and the proportion of cells positive for B7-H3 CAR were measured by FACS staining. After the CAR T cells expressing different B7-H3 scFv were incubated with 0. mu.g/ml, 0.25. mu.g/ml, 0.5. mu.g/ml and 1. mu.g/ml of 2Ig-B7-H3-Fc recombinant protein (FIG. 5) or only with 1. mu.g/ml of 2Ig-B7-H3-Fc recombinant protein (FIGS. 6, 8 and 11) for 30 minutes, the cells were incubated with PE-anti-human IgG Fc antibody for staining, and the staining results were examined by flow cytometry. C503 is a published anti-B7-H3 scFv as a positive control. NTD is a control T cell that is not transfected.

As shown in FIG. 5, CAR T cells expressing different anti-B7-H3 scFv bound to 2Ig-B7-H3-Fc recombinant protein to different extents, and the extent of binding correlated with the concentration and affinity of 2Ig-B7-H3-Fc recombinant protein.

As shown in figures 6, 8, 11, different B7-H3 CAR mRNA electroporated T cells expressed different proportions of CAR cells, with a higher proportion of CAR positive cells for both CAR-574 and C503T cell populations.

Example 5 preparation and characterization of Jeko1 cell line expressing varying amounts of B7-H3 protein levels

Electroporation of1X 10 with different amounts (0. mu.g, 0.4. mu.g, 2. mu.g and 10. mu.g) of B7-H3 mRNA⁷ Jeko1 tumor cells. The electroporation process was the same as above, with only the parameters changed, using 300V, 0.5 ms. FACS staining of varying amounts of B7-H3 mRNA electroporated Jeko1 cells by APC-isotype antibody and APC-anti-human B7-H3 antibody was used to measure B7-H3 expression in Jeko1 tumor cells. As shown in FIG. 10, Jeko1 cells did not express endogenous B7-H3, and the ectopic expression level of B7-H3 correlated with the amount of B7-H3 mRNA electroporated into Jeko1 cells.

Example 6 in vitro cytotoxicity assay

Cytotoxicity of B7-H3 CAR T cells against tumor cells was measured in an in vitro cytotoxicity assay. EGFP-expressing tumor cells or EGFP-Jeko1 cells electroporated with varying amounts of B7-H3 mRNA were seeded at 10000 cells/100 ul/well in flat-bottom 96-well plates; CAR T cells were diluted to appropriate concentration and co-cultured with tumor cells at a 1: 1E/T ratio, then the plates were placed into the IncuCyte S3 machine and the scan parameters set. After scanning for 3 days, analyzing the integral intensity of the total green cells (GCU x mum/hole) or the increase multiple of the integral intensity of the total green cells to calculate the killing efficiency.

FIG. 7 shows the killing curves of mRNA-based anti-B7-H3 CAR T cells against SKOV3-CBG, A549-CBG, Caski-CBG tumor cells in the first killing experiment at an E/T ratio of 1:1 (C503 is a positive control). As shown, CAR T cells expressing CAR-574, -C503 blocked growth of the three B7-H3 positive tumor cells to varying degrees, even eliminating tumor cells, suggesting that these scFv-based CAR T cells are highly cytotoxic to tumor cells.

FIG. 9 shows the killing curves of mRNA-based anti-B7-H3 CAR T cells at an E/T ratio of 1:1 against tumor cells endogenously expressing and not expressing B7-H3 in a second killing experiment. As shown, CAR-574, -C503 expressing CAR T cells effectively prevented the growth of endogenous high level B7-H3 expressing tumor cells, and CAR-574 CAR T cells had no inhibitory effect on the growth of endogenously non-expressing and very low B7-H3 expressing tumor cells.

FIG. 13 shows killing curves for different mRNA-based anti-B7-H3 CAR T cells in a third killing experiment at an E/T ratio of 1:1 against tumor cells endogenously not expressing and expressing B7-H3 and against Jeko1 cells exogenously expressing B7-H3 at different levels. As shown in the figure, the CAR T cell expressing CAR-574 effectively prevented the growth of tumor cells expressing B7-H3 at high level, and had no inhibition on the growth of tumor cells not expressing B7-H3, and had low or no inhibition on tumor cells expressing B7-H3 at very low level, confirming that it killed tumor cells expressing B7-H3 at high level and prevented the damage to normal tissues expressing B7-H3 at very low level.

Example 7 CAR T cell CD107a staining

CD107a is an early activation marker for T cells. Activation of B7-H3 CAR T cells by Jeko1 tumor cells expressing different levels of B7-H3 was measured using CD107a staining by the following steps: add 20 μ l PE-CD107a mAb to each well of a 96-well plate; tumor cells were diluted to1X 10⁶ Perml, and seeded on 96-well disks (100. mu.l/well); dilution of CAR T cells to1X 10⁶ Per ml, seeded in 96-well round plates (100. mu.l/well); the plate was centrifuged at 500 rpm × 5 minutes to make the cells well adhered, and cultured at 37 ℃ for 1 hour; golgi stop was added to each well (20 μ l/well) after being diluted 1500-fold with medium; the cells were further cultured at 37 ℃ for 2.5 hours, stained with anti-CD 3-APC and anti-CD 8-FITC antibodies at 37 ℃ for 30 minutes, washed and analyzed by flow cytometry.

In this study, the activation of B7-H3 CAR T cells (CAR-574) by B7-H3 mRNA electroporated Jeko1 tumor cells was measured by CD107a staining (0 μ g, 0.4 μ g, 2 μ g, and 10 μ g). As shown in figure 12, CAR T cells can be specifically activated by Jeko1 cells transfected with B7-H3, but not by Jeko1 cells that were not transfected with B7-H3. These results indicate that B7-H3-expressing tumor cells can activate the above B7-H3 CAR T cells.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> Shanghai excellent Biopharmaceutical Co., Ltd

<120> anti-B7-H3 antibodies and uses thereof

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgcagctggc acgacaggtt tc 22

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgtcagactg tagcacgtt 19

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aacaattgaa ttcaggagga 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cctcctaaga agcgtagtc 19

<210> 5

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Tyr Tyr Met Ser

1 5

<210> 6

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Tyr Ile Ser Ser Gly Ser Asn Ile Tyr Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 7

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Arg Arg Ser Ser Ser Gly Trp Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 8

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Asp Ala Ser Thr Leu Glu Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Gln Phe Asn Ser Phe Pro Leu Thr

1 5

<210> 11

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser

20 25 30

<210> 12

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 13

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 15

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 16

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Asn Leu Leu Ile Tyr

1 5 10 15

<210> 17

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 18

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly

1 5 10

<210> 19

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Asn Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Asn Ile Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Arg Ser Ser Ser Gly Trp Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 21

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gccatccggt tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcatcagc agtgctttag cctggtatca gcagaaacca 120

gggaaacctc ctaacctcct gatctatgat gcctccactt tggaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag tttaatagtt tccctctcac tttcggcgga 300

gggaccaagc tggagatcaa a 321

<210> 22

<211> 366

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gaggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggaat aaccttcagt gactactaca tgagctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagtg gtagtaatat atactacgca 180

gactcagtga agggccgatt caccatctcc agagacaacg ccaagaactc actgtatctg 240

caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aaggcggtct 300

agcagtggct ggtactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcctca 366

<210> 23

<211> 244

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Asn Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

115 120 125

Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Ile Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Gly

165 170 175

Ser Asn Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Arg Ser Ser Ser

210 215 220

Gly Trp Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser

<210> 24

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 25

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 26

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 27

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 28

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgacg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 29

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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> 30

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 31

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 32

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 33

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 34

<211> 507

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa tagtttccct 60

ctcactttcg gcggagggac caagctggag atcaaaggtg gtggtggttc tggcggcggc 120

ggctccggag gtggtggatc cgaggtgcag ctggtggagt ctgggggagg cttggtcaag 180

cctggagggt ccctgagact ctcctgtgca gcctctggaa taaccttcag tgactactac 240

atgagctgga tccgccaggc tccagggaag gggctggagt gggtttcata cattagtagt 300

ggtagtaata tatactacgc agactcagtg aagggccgat tcaccatctc cagagacaac 360

gccaagaact cactgtatct gcaaatgaac agcctgagag ccgaggacac ggctgtgtat 420

tactgtgcga gaaggcggtc tagcagtggc tggtactact acggtatgga cgtctggggc 480

caagggacca cggtcaccgt ctcctca 507

<210> 35

<211> 5105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 60

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 120

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 180

atgaccatga ttacgccaag ctctaatacg actcactata ggggagaccc tcgaccgtcg 240

attgtccact ggtcaacaat agatgactta caactaatcg gaaggtgcag agactcgacg 300

ggagctaccc taacgtcaag acgagggtaa agagagagtc caattctcaa agccaatagg 360

cagtagcgaa agctgcaaga gaatgaaaat ccgttgacct taaacggtcg tgtgggttca 420

agtccctcca cccccacgcc ggaaacgcaa tagccgaaaa acaaaaaaca aaaaaaacaa 480

aaaaaaaacc aaaaaaacaa aacacattaa aacagcctgt gggttgatcc cacccacagg 540

cccattgggc gctagcactc tggtatcacg gtacctttgt gcgcctgttt tataccccct 600

cccccaactg taacttagaa gtaacacaca ccgatcaaca gtcagcgtgg cacaccagcc 660

acgttttgat caagcacttc tgttaccccg gactgagtat caatagactg ctcacgcggt 720

tgaaggagaa agcgttcgtt atccggccaa ctacttcgaa aaacctagta acaccgtgga 780

agttgcagag tgtttcgctc agcactaccc cagtgtagat caggtcgatg agtcaccgca 840

ttccccacgg gcgaccgtgg cggtggctgc gttggcggcc tgcccatggg gaaacccatg 900

ggacgctcta atacagacat ggtgcgaaga gtctattgag ctagttggta gtcctccggc 960

ccctgaatgc ggctaatcct aactgcggag cacacaccct caagccagag ggcagtgtgt 1020

cgtaacgggc aactctgcag cggaaccgac tactttgggt gtccgtgttt cattttattc 1080

ctatactggc tgcttatggt gacaattgag agatcgttac catatagcta ttggattggc 1140

catccggtga ctaatagagc tattatatat ccctttgttg ggtttatacc acttagcttg 1200

aaagaggtta aaacattaca attcattgtt aagttgaata cagcaaatct agagccacca 1260

tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac gccgccaggc 1320

cgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnaccacgac gccagcgccg 1440

cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1500

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 1560

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1620

ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1680

ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1740

ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1800

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgacgttttg 1860

gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1920

gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1980

atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 2040

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaagtcgac 2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa caaaaaacaa aacggctatt atgcgttacc ggcgagacgc tacggactta 2220

aataattgag ccttaaagaa gaaattcttt aagtggatgc tctcaaactc agggaaacct 2280

aaatctagtt atagacaagg caatcctgag ccaagccgaa gtagtaatta gtaagaccag 2340

tggacaatcg acggataaca gcatatctag caaagaagag cactagtggc gcctgatgcg 2400

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataggccgct gtattctata 2460

gtgtcaccta aatggccgca caattcactg gccgtcgttt tacaacgtcg tgactgggaa 2520

aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 2580

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 2640

tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 2700

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 2760

agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 2820

ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 2880

cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 2940

gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 3000

aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 3060

ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa 3120

atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3180

tgagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 3240

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 3300

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaacaata 3360

aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa 3420

acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 3480

tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc 3540

gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 3600

gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 3660

atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc 3720

caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 3780

ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 3840

cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 3900

gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca 3960

ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 4020

gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 4080

gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 4140

tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 4200

gatgagtttt tctaagaatt aattcatgac caaaatccct taacgtgagt tttcgttcca 4260

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 4320

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 4380

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 4440

tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 4500

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 4560

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 4620

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 4680

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 4740

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 4800

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 4860

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 4920

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 4980

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 5040

cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 5100

gcgtt 5105

<210> 36

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ggggctgtct gtctgtctca t 21

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gtcctcagct cctgcattct c 21

<210> 38

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

catgtacgtt gctatccagg c 21

<210> 39

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ctccttaatg tcacgcacga t 21

Claims (19)

1. An anti-B7-H3 antibody, wherein the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, and the anti-B7-H3 antibody has the following technical characteristics:

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence shown in SEQ ID No. 5;

<2> the heavy chain variable region comprises CDR-H2 having an amino acid sequence shown in SEQ ID No. 6;

<3> the heavy chain variable region comprises CDR-H3 having an amino acid sequence shown in SEQ ID No. 7;

<4> the variable region of the light chain comprises CDR-L1 having an amino acid sequence shown in SEQ ID No. 8;

<5> the variable region of the light chain comprises CDR-L2 having an amino acid sequence shown in SEQ ID No. 9;

<6> the variable region of the light chain comprises CDR-L3 having an amino acid sequence shown in SEQ ID No. 10.

2. The anti-B7-H3 antibody of claim 1, wherein the amino acid sequences of the light chain variable region and the heavy chain variable region of the anti-B7-H3 antibody are shown as SEQ ID No.19 and SEQ ID No.20, respectively.

3. The anti-B7-H3 antibody of claim 1, wherein the anti-B7-H3 antibody is a single chain antibody.

4. The anti-B7-H3 antibody of claim 3, wherein the amino acid sequence of the anti-B7-H3 single chain antibody is set forth in SEQ ID No. 23.

5. Use of the anti-B7-H3 antibody of any one of claims 1-4 in the preparation or screening of a medicament for the treatment of a tumor, or in the preparation of a medicament for the diagnosis of a tumor.

6. Use according to claim 5, characterized in that the tumour therapeutic agent is a chimeric antigen receptor cell therapy agent.

7. An isolated polypeptide comprising a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the anti-B7-H3 antibody of any one of claims 1-4.

8. The isolated polypeptide of claim 7, wherein the polypeptide further comprises one or more of the following features:

1) the polypeptide is a chimeric antigen receptor;

2) the transmembrane domain is selected from CD8 a, CD28, DAP 10;

3) the endodomain comprises a costimulatory domain and/or a signaling domain;

4) the polypeptide comprises the anti-B7-H3 antibody, a transmembrane domain and an intracellular domain from the N end to the C end in sequence.

9. An isolated polynucleotide encoding the variable regions of the heavy and light chains of the anti-B7-H3 antibody of any one of claims 1-4, or encoding the polypeptide of claim 7 or 8.

10. A nucleic acid construct comprising the isolated polynucleotide of claim 9.

11. The nucleic acid construct of claim 10, wherein the nucleic acid construct is a viral vector or a non-viral vector.

12. The nucleic acid construct of claim 11, wherein the non-viral vector is an mRNA vector.

13. A cell expressing the polypeptide of claim 7 or 8.

14. The cell of claim 13, wherein the cell is a CAR T cell or a CAR NK cell.

15. A method for producing a cell according to any one of claims 13 to 14, comprising transferring the nucleic acid construct according to any one of claims 10 to 12 to a host cell.

16. The method of claim 15, wherein the method of transfer is selected from electroporation or viral transduction.

17. The method according to claim 15, wherein the host cell is a T cell or an NK cell.

18. A pharmaceutical composition comprising the anti-B7-H3 antibody or antigen-binding fragment thereof of any one of claims 1-4 and/or the cell of any one of claims 13-14, and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, further comprising one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a diagnostic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

Images