CN117024590A - Monoclonal antibody against human B7-H3 and application thereof - Google Patents
Monoclonal antibody against human B7-H3 and application thereof Download PDFInfo
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- CN117024590A CN117024590A CN202310873117.XA CN202310873117A CN117024590A CN 117024590 A CN117024590 A CN 117024590A CN 202310873117 A CN202310873117 A CN 202310873117A CN 117024590 A CN117024590 A CN 117024590A
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Abstract
The application discloses an anti-human B7-H3 monoclonal antibody and application thereof. In particular, the application discloses a novel B7-H3 targeted monoclonal antibody, particularly a single chain antibody. The application also discloses a method for preparing the monoclonal antibody. The monoclonal antibody provided by the application can be combined with B7-H3 antigen with high specificity, has high affinity and has obvious anti-tumor activity and the like.
Description
The application relates to a Chinese application division application with application number 2020103243477, application date of 2020, 4 months and 22 days, and the name of the monoclonal antibody against human B7-H3 and application thereof.
Technical Field
The application relates to biomedical or biopharmaceutical technology, in particular to monoclonal antibodies of human B7-H3 and derivatives thereof, such as single chain antibodies, bispecific antibodies, antibody coupling drugs, CAR-T cells and the like, and corresponding applications thereof.
Background
B7-H3 (also known as CD 276) is a type I transmembrane protein belonging to the B7 immunoglobulin superfamily. In humans, mRNA of B7-H3 is widely expressed in various normal tissues, but protein is not expressed or is expressed under in normal tissues. The B7-H3 protein is widely expressed in various cancer tissues such as lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer, etc., and high expression of the B7-H3 protein is associated with disease progression and poor prognosis of various cancers. In addition, in the tumor microenvironment, B7-H3 is also overexpressed on tumor-associated vascular endothelium and fibroblasts, but not on normal vascular endothelium as well as physiological angiogenic endothelial cells. Given the property that B7-H3 is not expressed or is expressed in low levels in normal tissues but is highly expressed in various cancer tissues, B7-H3 is a tumor-associated antigen with great potential.
Thus, there is an urgent need in the art to develop antibodies that bind B7-H3 with high affinity and specificity.
Disclosure of Invention
The object of the present invention is to provide antibodies that bind B7-H3 with high affinity and specificity.
In a first aspect of the invention there is provided an antibody against B7-H3, said antibody having a heavy chain variable region and a light chain variable region,
the heavy chain variable region has 3 complementarity determining regions VH-CDRs, the 3 VH-CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID No.8n +2,
VH-CDR2 shown in SEQ ID No.8n +3, and
VH-CDR3 shown in SEQ ID No.8n +4;
wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34;
and/or the light chain variable region has 3 complementarity determining regions VL-CDRs, the 3 VL-CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID No.8n +6,
VL-CDR2 shown in SEQ ID No.8n +7, and
VL-CDR3 as shown in SEQ ID No.8n +8;
wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34;
Wherein any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one amino acid and which is capable of retaining its B7-H3 binding affinity.
In another preferred embodiment, the heavy chain of the antibody has the amino acid sequence of any one of SEQ ID nos. 8n +1, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
In another preferred embodiment, the light chain of the antibody has the amino acid sequence of any one of SEQ ID nos. 8n +5, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
In another preferred embodiment, n is any integer from 0 to 8.
In another preferred embodiment, n is any integer from 9 to 16.
In another preferred embodiment, the amino acid sequence of any one of the CDRs comprises a derivative CDR sequence of 1, 2 or 3 amino acids, which is added, deleted, modified and/or substituted, and such that a derivative antibody comprising VH and VL comprising said derivative CDR sequence retains affinity for binding to B7-H3.
In another preferred embodiment, the ratio of the affinity F1 for binding B7-H3 of the derivatized antibody to the affinity F0 for binding B7-H3 of the corresponding non-derivatized antibody (F1/F0) is from 0.5 to 2, preferably from 0.7 to 1.5, and more preferably from 0.8 to 1.2.
In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted is 1 to 5 (e.g., 1 to 3, preferably 1 to 2, more preferably 1).
In another preferred embodiment, the derivative sequence which has been added, deleted, modified and/or substituted with at least one amino acid and which retains the binding affinity for B7-H3 is an amino acid sequence having at least 95% homology or sequence identity.
In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.
In another preferred embodiment, the heavy chain constant region is of human origin and/or the light chain constant region is of human origin.
In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region and the light chain constant region is a human antibody light chain kappa constant region.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.
In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:
(a) Inhibit tumor cell migration or metastasis;
(b) Inhibit tumor growth.
In another preferred embodiment, the antibody is a single chain antibody (scFV).
In another preferred embodiment, the antibody has a structure represented by formula I or II:
Z1-L1-Z2-Z3(I)
Z2-L1-Z1-Z3(II)
in the method, in the process of the invention,
z1 is a heavy chain variable region VH;
z2 is the light chain variable region VL;
l1 is none or a peptide linker;
z3 is an antibody constant region (Fc region);
each "-" represents a peptide bond.
In another preferred embodiment, L1 is a flexible peptide linker, preferably (G 4 S) 3 。
In another preferred embodiment, the linker fragment (linker) of the scFv is GSTSGSGKPGSGEGS or GKPGSGKPGSGKPGSGKPGS.
In another preferred embodiment, Z3 is the Fc of IgG, preferably the Fc of IgG 1.
In another preferred embodiment, Z3 is the Fc of a human antibody.
In another preferred embodiment, the antibody has a structure represented by formula III:
VH-(G 4 S) 3 -VL-huIgG1Fc(III)
in the method, in the process of the invention,
VH is a heavy chain variable region;
VL is the light chain variable region;
(G 4 S) 3 is a peptide linker;
huIgG1Fc is the constant region of human IgG1 antibodies.
In another preferred embodiment, the VH, VL and sequence numbers of the respective CDRs of the antibody are as shown in table a:
table A sequence numbers of VH, VL and respective CDRs of antibody clones (SEQ ID No:)
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a Framework Region (FR) and the light chain variable region of the antibody further comprises a Framework Region (FR).
In another preferred example, the amino acid sequence in the framework region of the VH and/or the framework region of the VL may comprise a derivative sequence optionally having at least one amino acid added, deleted, modified and/or substituted and capable of retaining its B7-H3 binding affinity.
In another preferred embodiment, said heavy chain variable region and said light chain variable region comprise CDRs selected from the group consisting of:
Wherein any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted for at least one amino acid and which is capable of retaining B7-H3 binding affinity.
In another preferred embodiment, the antibody is selected from the group consisting of:
in another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID No.1, 9, 17, 25, 33, 41, 49, 57, 65.
In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID No.5, 13, 21, 29, 37, 45, 53, 61, 69.
In a second aspect of the present invention, there is provided a recombinant protein comprising:
(i) An antibody according to the first aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the recombinant protein is a fusion protein.
In another preferred embodiment, the recombinant protein is a membrane protein.
In another preferred embodiment, the recombinant protein is a Chimeric Antigen Receptor (CAR).
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a third aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) An antibody according to the first aspect; and
(2) The recombinant protein of the second aspect.
In a fourth aspect of the invention, there is provided a vector comprising a polynucleotide according to the third aspect.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a fifth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fourth aspect or a polynucleotide according to the third aspect integrated into the genome.
In another preferred embodiment, the host cell comprises a human cell.
In another preferred embodiment, the host cell comprises an immune cell.
In a sixth aspect of the invention, there is provided an antibody conjugate comprising:
(a) An antibody moiety, said antibody being as described in the first aspect, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In a seventh aspect of the invention, there is provided an immune cell expressing or exposed outside the cell membrane the antibody of the first aspect and a fusion protein comprising the antibody.
In another preferred embodiment, the immune cells include NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In another preferred embodiment, the fusion protein is a Chimeric Antigen Receptor (CAR).
In an eighth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition comprises (i) 0.01 to 99.99wt% of the antibody according to the first aspect, the recombinant protein according to the second aspect, the antibody conjugate according to the sixth aspect, the immune cell according to the seventh aspect, or a combination thereof, and (ii) 0.01 to 99.99wt% of a pharmaceutically acceptable carrier, said percentages being weight percentages of the pharmaceutical composition.
In a ninth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof, wherein the active ingredient is used to (a) prepare a diagnostic reagent or kit; and/or (B) preparing a medicament for preventing and/or treating diseases related to B7-H3 expression or dysfunction.
In another preferred embodiment, the diagnostic reagent is a test strip or a test plate.
In another preferred embodiment, the disorder associated with B7-H3 expression or dysfunction is selected from the group consisting of: cancer, autoimmune diseases.
In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, prostate cancer, breast cancer, colorectal cancer, renal cancer, ovarian cancer, and liver cancer.
In another preferred embodiment, the diagnostic reagent or kit is for:
(1) Detecting B7-H3 protein in the sample; and/or
(2) Detecting endogenous B7-H3 protein in tumor cells; and/or
(3) Detecting the tumor cells expressing the B7-H3 protein.
In another preferred embodiment, the medicament is for preventing and/or treating a disease associated with B7-H3 expression or dysfunction selected from the group consisting of: cancer, autoimmune disease.
In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, prostate cancer, breast cancer, colorectal cancer, renal cancer, ovarian cancer, and liver cancer.
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In another preferred embodiment, the diagnostic reagent or kit is used for diagnosing B7-H3 related diseases.
In another preferred embodiment, the diagnostic reagent or kit is used to detect B7-H3 protein in a sample.
In a tenth aspect of the invention, there is provided a method of detecting B7-H3 protein in an in vitro sample (including diagnostic or non-diagnostic), the method comprising the steps of:
(1) Contacting the sample with an antibody according to the first aspect of the invention in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of B7-H3 protein in the sample.
In an eleventh aspect of the present invention, there is provided a detection plate comprising: a substrate (support plate) and a test strip comprising an antibody according to the first aspect of the invention.
In a twelfth aspect of the invention, there is provided a kit comprising:
(1) A first container comprising an antibody according to the first aspect of the invention; and/or
(2) A second container comprising a second antibody against the antibody of the first aspect of the invention;
or,
the kit comprises the detection plate.
In a thirteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.
In a fourteenth aspect of the present invention, there is provided a pharmaceutical combination comprising:
(i) A first active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof;
(ii) A second active ingredient comprising a second antibody, or a chemotherapeutic agent.
In another preferred embodiment, the second antibody is selected from the group consisting of: CTLA4 antibody, PD-1 antibody, PD-L1 antibody, 4-1BB antibody.
In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: docetaxel, carboplatin, or a combination thereof.
In a fifteenth aspect of the present invention there is provided a method of treating a disease associated with B7-H3 expression or dysfunction comprising the steps of: administering to a subject in need thereof an effective amount of an antibody according to the first aspect, a recombinant protein according to the second aspect, an antibody conjugate according to the sixth aspect, an immune cell according to the seventh aspect, or a combination thereof.
In another preferred embodiment, the disorder associated with B7-H3 expression or dysfunction is selected from the group consisting of: cancer, autoimmune diseases.
In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, prostate cancer, breast cancer, colorectal cancer, renal cancer, ovarian cancer, liver cancer, and the like.
In another preferred embodiment, the method further comprises: a safe and effective amount of a second antibody is administered to the subject before, during and/or after administration of the first active ingredient.
In another preferred embodiment, the second antibody is selected from the group consisting of: PD-1 antibody, CTLA4 antibody, PD-L1 antibody, 4-1BB antibody.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the results of screening of embryonic kidney cell HEK 293T cell lines (designated B cells) over-expressed in human 4 Ig-B7-H3.
FIG. 2 shows the results of screening of the embryonic kidney cell HEK 293T cell line (designated C-cell) for overexpression of mouse 2 Ig-B7-H3.
FIG. 3 shows SDS-PAGE of recombinant proteins (extracellular domain of B7-H3 protein), wherein lanes B7-H3ECD (29-465) -6His (R) represent a reductive sample of recombinantly expressed human B7-H3 extracellular domain protein (with a 6His tag); lanes B7-H3ECD (29-465) -6His (NR) represent non-reducing samples of recombinantly expressed human B7-H3 extracellular proteins (with a 6His tag).
FIG. 4 shows a SEC-HPLC plot of recombinant protein (extracellular domain of B7-H3 protein).
FIG. 5 shows the CDR1 amino acid sequence alignment of a portion of scFv single chain antibody VH.
FIG. 6 shows the CDR2 amino acid sequence alignment of a portion of scFv single chain antibody VH.
FIG. 7 shows the CDR3 amino acid sequence alignment of a portion of scFv single chain antibody VH.
FIG. 8 shows the CDR1 amino acid sequence alignment of a portion of scFv single chain antibody VL.
FIG. 9 shows the CDR2 amino acid sequence alignment of a portion of scFv single chain antibody VL.
FIG. 10 shows the CDR3 amino acid sequence alignment of a portion of scFv single chain antibody VL.
FIG. 11 shows the affinity curves and IC50 values of a portion of scFv single chain antibodies and human 4Ig-B7-H3 ECD protein detected by enzyme-linked immunosorbent assay (ELISA). Wherein, a to I of fig. 11 show the detection results of different antibodies, respectively.
FIG. 12 shows the binding of a portion of scFv single chain antibodies to A, B, C cells detected by flow cytometry. Wherein, the binding conditions of the following single-chain antibodies are included from top to bottom.
(A) 96-3,96-14,96-2,96-8,90-26,90-16,81A1,81A15 single chain antibody;
(B) 81A55,81A9,81A58,81A68,81A53,81A40,81A14,81A5 single chain antibody;
(C) 81A37,81A2,81A57,81A30,81A21,81A18,81A31,81A19 single chain antibody;
(D) 81A6,81A8,81A71,81A56,81A66,81A70 single chain antibody;
(E) 81A22,81A4,81A25,81A24,81A34 single chain antibody.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies to unexpectedly obtain a group of B7-H3 antibodies having a novel amino acid sequence by mass screening using a humanized B7-H3 protein and a cell line stably expressing the B7-H3 protein as immunogens. The B7-H3 antibody provided by the invention is a B7-H3 antibody with excellent biological characteristics, and has high affinity and specificity to human B7-H3 protein, so that the antibody can be applied to treatment of tumors and other related diseases. The present invention has been completed on the basis of this finding.
Terminology
In the present invention, "VH-CDR1" is used interchangeably with "CDR-H1" and both refer to CDR1 of the heavy chain variable region; "VH-CDR2" is used interchangeably with "CDR-H2" and refers to CDR2 of the heavy chain variable region; "VH-CDR3" is used interchangeably with "CDR-H3" and refers to CDR3 of the heavy chain variable region. "VL-CDR1" is used interchangeably with "CDR-L1" and refers to CDR1 of the light chain variable region; "VL-CDR2" is used interchangeably with "CDR-L2" and refers to the CDR2 of the variable region of the light chain; "VL-CDR3" is used interchangeably with "CDR-L3" and refers to the CDR3 of the variable region of the light chain.
The term "about" may refer to a value or composition that is within an acceptable error of a 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 measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a well known method to those skilled in the art.
B7-H3
B7-H3 (also known as CD 276) is a type I transmembrane protein belonging to the B7 immunoglobulin superfamily, B7-H3 having 20-27% homology with other family members. In mice, the B7-H3 gene is located on mouse chromosome 9, and the extracellular structure of its protein consists of one IgV and one IgC (VC, 2 Ig-B7-H3); in humans, the B7-H3 gene is located on human chromosome 15, and the human B7-H3 protein exists in two forms, one consisting of one IgV and IgC (VC, 2 Ig-B7-H3), the other in the form of two pairs of VCs in tandem (VCVC, 4 Ig-B7-H3), and the latter in the form most widely expressed in humans.
In humans, B7-H3 mRNA is widely expressed in a variety of normal tissues, but protein expression is severely limited and either not expressed or under expressed in normal tissues. The B7-H3 protein is widely expressed in various cancer tissues such as lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer, etc., and high expression of the B7-H3 protein is associated with disease progression and poor prognosis of various cancers. In addition, in the tumor microenvironment, B7-H3 is also overexpressed on tumor-associated vascular endothelium and fibroblasts, but not on normal vascular endothelium as well as physiological angiogenic endothelial cells. In addition, B7-H3 was found to exert a co-inhibitory effect in immunomodulation, which is capable of inhibiting T cell division by modulating NFAT, nfkb and AP-1 signaling pathways, and in B7-H3 deficient mice Th cells differentiate more into Th1 cells than Th2 cells, resulting in more severe airway inflammation.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
Single chain antibodies (scFv, single chain fragment variable) are antibodies composed of the variable regions (V) H And V L ) Is composed of a size of about 30kDa. Typically, at V H And V L Are linked by a 15-20 amino acid variable short peptide linker (linker) which is usually composed of a hydrophobic sequence, the most common design being (G) consisting of glycine (G) and serine (S) 4 S) 3 A linker. Compared with full-length antibodies, single-chain antibodies have smaller molecular weight while retaining binding specificity and affinity, and can better and faster infiltrate into tumor tissue or other tissues, while single-chain antibodies have shorter half-life in vivo and can be more rapidly excreted from the blood through the kidney, so that the exposure time of drugs or radionuclide-linked single-chain antibodies in normal tissues can be reduced. Furthermore, single chain antibodies can be used to construct affinity origins for CAR-T cells, have been widely used, and demonstrate their efficacy in clinical applications.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding capacity and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding portion of the antibody (see Kabat et al, NIHPubl. NoNO.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the anti-B7-H3 protein antibodies or fragments thereof.
As used herein, the terms "heavy chain variable region" and "V H "interchangeably used.
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to a B7-H3 protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.
The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
The antigen binding properties of antibodies can be described by 3 specific regions located in the heavy chain variable region, called variable regions (CDRs), which are separated into 4 Framework Regions (FR), the amino acid sequences of 4 FR being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having a B7-H3 protein binding activity and comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more amino acids, and addition of one or several amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the DNA encoding the antibodies of the invention under conditions of high or low stringency, and polypeptides or proteins obtained using antisera of the antibodies of the invention.
In the present invention, the antibodies may be monospecific, bispecific, trispecific, or multispecific.
The invention also provides other polypeptides, such as fusion proteins comprising a single domain antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the single domain antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 70 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.
TABLE 1
Initial residues | Representative substitution | Preferred substitution |
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 |
In the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids of the original amino acid sequence.
In the present invention, more preferably, the number of the added, deleted, modified and/or substituted amino acids may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, still more preferably 1 to 2.
Nucleic acid
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
Preparation method
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification, recombinant or artificial synthesis. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
In addition, the coding sequence of the antibody and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a genetically engineered vector, transferring it into cells, and isolating the relevant sequences from the propagated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
Vectors and host cells
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.
Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimides, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorinated); halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodine, bromine, or chlorine); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-di- (mercuromethyl) dioxane, while the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.
The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxazolodiazenes)) and vinca alkaloids (vinca alkaloids).
In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.
The invention also provides a method of making an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise: the antibody is bound to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is conjugated to the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody through the linker.
In some embodiments, the antibody drug conjugate ADC is of the formula:
wherein:
ab is an antibody that is conjugated to a polypeptide,
LU is the linker;
d is a drug;
and subscript p is a value selected from 1 to 8.
Application of
The invention also provides uses of the antibodies, antibody conjugate ADCs, recombinant proteins, and/or immune cells of the invention, e.g., for the preparation of diagnostic formulations or for the preparation of medicaments.
Preferably, the medicament is a medicament for preventing and/or treating diseases related to B7-H3 expression or dysfunction.
In the present invention, the diseases associated with B7-H3 expression or dysfunction are diseases associated with B7-H3 expression or dysfunction, which are conventional in the art. Preferably, the disease associated with B7-H3 expression or dysfunction is cancer.
In the present invention, the cancer is a cancer conventional in the art, preferably lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer and liver cancer.
The use of antibodies, ADCs, recombinant proteins, and/or immune cells of the invention include (but are not limited to):
(i) Diagnosing, preventing and/or treating tumorigenesis, growth and/or metastasis, in particular tumors with high B7-H3 expression. The tumors include (but are not limited to): lung cancer, prostate cancer, breast cancer, colorectal cancer, renal cancer, ovarian cancer, and liver cancer.
Detection application and kit
The antibodies of the invention, or ADCs thereof, may be used in detection applications, for example, for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include, for example, resected samples of tumors, tissue samples prepared by endoscopic methods or puncture of organs or needle biopsies.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated.
The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the route of administration of the pharmaceutical compositions of the present invention is preferably injection or oral. The administration by injection preferably comprises intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical compositions are in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be in the form of aqueous solutions, non-aqueous solutions or suspensions, more preferably tablets, capsules, granules, injections or infusions, etc.
The antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical composition is used for preventing and/or treating diseases related to B7-H3 expression or dysfunction.
The pharmaceutical composition of the invention can be directly used for combining B7-H3 protein molecules, thus being used for preventing and treating diseases such as tumors and the like.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the present invention, the pharmaceutical composition of the present invention preferably further comprises one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the protein and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.
In the present invention, the pharmaceutical composition is preferably administered in an amount effective to reduce or delay the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the symptoms to be treated and the consideration of the results sought. The skilled artisan can determine the effective amount by using the factors described above on an individual basis and the like and using no more than routine experimentation.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The invention provides application of the pharmaceutical composition in preparing medicines for preventing and/or treating diseases related to B7-H3 expression or dysfunction. Preferably, the disease associated with B7-H3 expression or dysfunction is cancer, autoimmune disease.
Methods and compositions for detecting B7-H3 protein in a sample
The invention also provides a method of detecting a B7-H3 protein (e.g., detecting an over-expressing B7-H3 cell) in a sample, comprising the steps of: the antibody is contacted with a sample to be detected in vitro, and whether the antibody is combined with the sample to be detected to form an antigen-antibody complex is detected.
The meaning of overexpression is conventional in the art and refers to overexpression of the B7-H3 protein in the sample to be examined (altered by transcription, post-transcriptional processing, translation, post-translational processing and protein degradation), as well as to local overexpression and increased functional activity due to altered protein transport patterns (increased nuclear localization), as in the case of increased enzymatic hydrolysis of the substrate.
In the present invention, the above-mentioned detection mode of whether or not an antigen-antibody complex is formed by binding is a conventional detection mode in the art, preferably a flow cytometry (FACS) detection.
The invention provides a composition for detecting B7-H3 protein in a sample, which comprises the antibody, recombinant protein, antibody conjugate, immune cell or combination thereof as an active ingredient. Preferably, it further comprises a compound composed of the functional fragment of the above antibody as an active ingredient.
The main advantages of the invention include:
(a) The antibodies of the invention have high affinity for human B7-H3.
(b) The antibody of the invention has high specificity to human B7-H3.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
EXAMPLE 1 construction of human 4Ig-B7-H3 overexpressed embryonic kidney cell HEK 293T cell line (designated B cell)
Resuscitating HEK 293T cell strain with low generation number, and culturing at 37deg.C and 5% CO 2 Saturated steam, the medium was DMEM (Gibco) with 10% fbs (Gibco), to which was added 1% penicillin-streptomycin (Gibco). After 2-3 passages of culture, the cells were inoculated into 6-well plates and transfected until confluence of the monolayer cells was 70-90%. Mu.g of plasmid (expressing B7-H3 and eGFP fluorescent protein) per well was transfected with X-tremeGENE HP DNA Transfection Reagent (Roche). Transfected cells were prepared as single cell suspensions after 2-3 successive generations of culture, cells were seeded into 96-well plates at a density of 0.8 cells per well, and single clones were selected by observing their GFP fluorescence and expanded stepwise. And detecting GFP single peak condition by a flow cytometry in the process of expanding culture, removing multi-peak clone, and finally further verifying the obtained GFP single peak clone by a B7-H3 flow antibody to obtain the required positive clone.
The nucleotide gene sequence for expression of the 4Ig-B7-H3 protein is as follows:
ATGCTGCGTCGGCGGGGCAGCCCTGGCATGGGTGTGCATGTGGGTGCAGCCCTGGGAGCACTGTGGTTCTGC
CTCACAGGAGCCCTGGAGGTCCAGGTCCCTGAAGACCCAGTGGTGGCACTGGTGGGCACCGATGCCACCCTGTGCTG
CTCCTTCTCCCCTGAGCCTGGCTTCAGCCTGGCACAGCTCAACCTCATCTGGCAGCTGACAGATACCAAACAGCTGG
TGCACAGCTTTGCTGAGGGCCAGGACCAGGGCAGCGCCTATGCCAACCGCACGGCCCTCTTCCCGGACCTGCTGGCA
CAGGGCAACGCATCCCTGAGGCTGCAGCGCGTGCGTGTGGCGGACGAGGGCAGCTTCACCTGCTTCGTGAGCATCCG
GGATTTCGGCAGCGCTGCCGTCAGCCTGCAGGTGGCCGCTCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACA
AGGACCTGCGGCCAGGGGACACGGTGACCATCACGTGCTCCAGCTACCAGGGCTACCCTGAGGCTGAGGTGTTCTGG
CAGGATGGGCAGGGTGTGCCCCTGACTGGCAACGTGACCACGTCGCAGATGGCCAACGAGCAGGGCTTGTTTGATGT
GCACAGCATCCTGCGGGTGGTGCTGGGTGCAAATGGCACCTACAGCTGCCTGGTGCGCAACCCCGTGCTGCAGCAGG
ATGCGCACAGCTCTGTCACCATCACACCCCAGAGAAGCCCCACAGGAGCCGTGGAGGTCCAGGTCCCTGAGGACCCG
GTGGTGGCCCTAGTGGGCACCGATGCCACCCTGCGCTGCTCCTTCTCCCCCGAGCCTGGCTTCAGCCTGGCACAGCT
CAACCTCATCTGGCAGCTGACAGACACCAAACAGCTGGTGCACAGTTTCACCGAAGGCCGGGACCAGGGCAGCGCCT
ATGCCAACCGCACGGCCCTCTTCCCGGACCTGCTGGCACAAGGCAATGCATCCCTGAGGCTGCAGCGCGTGCGTGTG
GCGGACGAGGGCAGCTTCACCTGCTTCGTGAGCATCCGGGATTTCGGCAGCGCTGCCGTCAGCCTGCAGGTGGCCGC
TCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACAAGGACCTGCGGCCAGGGGACACGGTGACCATCACGTGCT
CCAGCTACCGGGGCTACCCTGAGGCTGAGGTGTTCTGGCAGGATGGGCAGGGTGTGCCCCTGACTGGCAACGTGACC
ACGTCGCAGATGGCCAACGAGCAGGGCTTGTTTGATGTGCACAGCGTCCTGCGGGTGGTGCTGGGTGCGAATGGCAC
CTACAGCTGCCTGGTGCGCAACCCCGTGCTGCAGCAGGATGCGCACGGCTCTGTCACCATCACAGGGCAGCCTATGA
CATTCCCCCCAGAGGCCCTGTGGGTGACCGTGGGGCTGTCTGTCTGTCTCATTGCACTGCTGGTGGCCCTGGCTTTC
GTGTGCTGGAGAAAGATCAAACAGAGCTGTGAGGAGGAGAATGCAGGAGCTGAGGACCAGGATGGGGAGGGAGAAGG
CTCCAAGACAGCCCTGCAGCCTCTGAAACACTCTGACAGCAAAGAAGATGATGGACAAGAAATAGCCTGA(SEQ ID
No:281)
experimental results:
the results are shown in FIG. 1, and based on the results of the flow analysis, 3E11 clones were finally selected as B cells for post-selection.
EXAMPLE 2 construction of mouse 2Ig-B7-H3 overexpressing embryonic kidney cell HEK 293T cell line (designated C cell)
Example 1 was repeated except that the coding sequence of human 4Ig-B7-H3 was replaced with the coding sequence of mouse 2 Ig-B7-H3.
The nucleotide gene sequence for expressing the mouse 2Ig-B7-H3 protein is as follows:
ATGCTTCGAGGATGGGGTGGCCCCAGTGTGGGTGTGTGTGTGCGCACAGCACTGGGGGTGCTGTGCCTCTGC
CTCACAGGAGCTGTGGAAGTCCAGGTCTCTGAAGACCCCGTGGTGGCCCTGGTGGACACGGATGCCACCCTACGCTG
CTCCTTTTCCCCAGAGCCTGGCTTCAGTCTGGCACAGCTCAACCTCATCTGGCAGCTGACAGACACCAAACAGCTGG
TGCACAGCTTCACGGAGGGCCGGGACCAAGGCAGTGCCTACTCCAACCGCACAGCGCTCTTCCCTGACCTGTTGGTG
CAAGGCAATGCGTCCTTGAGGCTGCAGCGCGTCCGAGTAACCGACGAGGGCAGCTACACCTGCTTTGTGAGCATCCA
GGACTTTGACAGCGCTGCTGTTAGCCTGCAGGTGGCCGCCCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACA
AGGACCTACGTCCAGGGAACATGGTGACCATCACGTGCTCTAGCTACCAGGGCTATCCGGAGGCCGAGGTGTTCTGG
AAGGATGGACAGGGAGTGCCCTTGACTGGCAATGTGACCACATCCCAGATGGCCAACGAGCGGGGCTTGTTCGATGT
TCACAGCGTGCTGAGGGTGGTGCTGGGTGCTAACGGCACCTACAGCTGCCTGGTACGCAACCCGGTGTTGCAGCAAG
ATGCTCACGGCTCAGTCACCATCACAGGGCAGCCCCTGACATTCCCCCCTGAGGCTCTGTGGGTAACCGTGGGGCTC
TCTGTCTGTCTTGTGGTACTACTGGTGGCCCTGGCTTTCGTGTGCTGGAGAAAGATCAAGCAGAGCTGCGAGGAGGA
GAATGCAGGTGCCGAGGACCAGGATGGAGATGGAGAAGGATCCAAGACAGCTCTACGGCCTCTGAAACCCTCTGAAA
ACAAAGAAGATGACGGACAAGAAATTGCTTGA(SEQ ID No:282)
the results are shown in FIG. 2, and based on the results of the flow analysis, the 2B3 clone was finally selected as the C cell for subsequent screening.
The A cell construction method was the same as B, C cells except that only the eGFP fluorescent protein was expressed using the plasmid, and no other gene of interest was used as a control for the screening process.
EXAMPLE 3 extraction of B7-H3 ECD protein
The extracellular domain (ECD, amino acids 29-465) recombinant protein of human 4Ig-B7-H3 was expressed using eukaryotic expression system HEK 293-6E (available from Thermo Fisher Scientific) and its sequence:
LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPE(SEQ ID No:283)
the recombinant protein (extracellular domain of B7-H3 protein) was obtained by nickel column affinity purification.
SDS-PAGE was identified as shown in FIG. 3, and SEC-HPLC was identified as shown in FIG. 4, and the results indicated that the purity of the recombinant protein was >95%.
EXAMPLE 4 selection of clones capable of binding to cells overexpressing 4Ig-B7-H3
The phage display technology is used to screen the fully human natural antibody library to obtain different Fab libraries.
Method one, screening from these by ELISA and flow cytometry, yields clones capable of binding B cells and/or C cells. Repeated clones were removed by sequencing alignment to obtain specific clones capable of binding B cells and/or C cells.
Screening a fully human natural antibody library through B7-H3 ECD protein, screening by adopting an immune tube solid-phase immunoaffinity and magnetic bead liquid-phase immunoaffinity method, and sequencing and comparing to remove repeated clones to obtain specific clones capable of combining B cells and/or C cells.
Rechecking and verifying the specific clone obtained by the cell screening by ELISA and flow cytometry, and obtaining 29 clones with unique sequences and capable of combining B cells and/or C cells after removing the clones with repeated sequences after combining sequencing and comparison; the library of fully human natural antibodies was screened by B7-H3 ECD proteins from which 9 clones with unique sequences capable of binding B cells and/or C cells were obtained.
Thus, by the above two different technical routes, after excluding 3 clones with sequence redundancy, a total of 35 positive antibody clones with sequence uniqueness capable of binding B cells were obtained; and wherein 30 antibody clones were able to bind both B cells and C cells.
EXAMPLE 5 construction of Single chain antibodies and affinity Property sequencing
5.1 construction of Single chain antibodies
The above-obtained antibody was subjected to the process of passing (G 4 S) 3 The linker fragments are joined together to construct a single chain antibody (single chain fragment variable, scFv) which is fused with a human IgG1-Fc fragment to construct a construct of the structural form VH- (G) 4 S) 3 -VL-huIgG1 Fc fusion protein. These 35 single chain antibodies were recombinantly expressed using 293T cells and purified to obtain these antibodies.
5.2 affinity test
Based on the A, B, C cells and B7-H3 ECD proteins, the 35 single-chain antibodies are rechecked by adopting flow cytometry and enzyme-linked immunosorbent assay (ELISA) experiments, and the affinity and the specificity of the single-chain antibodies for binding B7-H3 molecules are classified and sequenced.
5.3 results
The structures of the 35 single chain antibodies constructed are listed in Table 1.
TABLE 1
The corresponding amino acid sequences are shown in Table 2
TABLE 2 amino acid sequences of VH and VL
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The corresponding DNA sequences are shown in Table 3.
TABLE 3 nucleotide sequences of VH and VL
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The CDRs of the VH and VL of a portion of the antibodies are listed in Table 4, wherein the sequence alignment of the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3 of a portion of the antibody clone is shown in FIGS. 5-10.
TABLE 4 CDR amino acid sequences of partial antibodies
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The nucleotide sequences of the CDRs of VH and VL of a portion of the antibodies are shown in table 5.
TABLE 5 nucleotide sequences of CDRs of partial antibodies
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The affinity of the partial antibodies was determined as shown in table 6, fig. 11 and fig. 12.
TABLE 6 affinity of partial scFv Single chain antibodies
As shown in FIG. 11, the enzyme-linked immunosorbent assay (ELISA) shows that the scFv single chain antibody and the human 4Ig-B7-H3ECD protein have excellent affinity and EC50 value.
As shown in FIG. 12, the flow cytometry detection shows that the scFv single-chain antibody of the application can specifically bind to human B7-H3ECD protein and can specifically recognize murine B7-H3 (the sequence and the structure of the scFv single-chain antibody are highly conserved with human B7-H3).
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. An antibody against B7-H3, wherein the antibody has a heavy chain variable region and a light chain variable region,
the heavy chain variable region has 3 complementarity determining regions VH-CDRs, the 3 VH-CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID No.8n +2,
VH-CDR2 shown in SEQ ID No.8n +3, and
VH-CDR3 shown in SEQ ID No.8n +4;
wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34;
And/or the light chain variable region has 3 complementarity determining regions VL-CDRs, the 3 VL-CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID No.8n +6,
VL-CDR2 shown in SEQ ID No.8n +7, and
VL-CDR3 as shown in SEQ ID No.8n +8;
wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34;
wherein any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one amino acid and which is capable of retaining its B7-H3 binding affinity.
2. The antibody of claim 1, wherein the heavy chain of the antibody has an amino acid sequence of any one of SEQ ID nos. 8n +1, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
3. The antibody of claim 1, wherein the light chain of the antibody has an amino acid sequence of any one of SEQ ID nos. 8n +5, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
4. The antibody of claim 1, wherein said antibody is a single chain antibody (scFV).
5. The antibody of claim 1, wherein the antibody has a structure according to formula I or II:
Z1-L1-Z2-Z3(I)
Z2-L1-Z1-Z3(II)
in the method, in the process of the invention,
z1 is a heavy chain variable region VH;
z2 is the light chain variable region VL;
l1 is none or a peptide linker;
z3 is an antibody constant region (Fc region);
each "-" represents a peptide bond.
6. The antibody of claim 1, wherein the antibody has a structure according to formula III:
VH-(G 4 S) 3 -VL-huIgG1Fc(III)
in the method, in the process of the invention,
VH is a heavy chain variable region;
VL is the light chain variable region;
(G 4 S) 3 is a peptide linker;
huIgG1Fc is the constant region of human IgG1 antibodies.
7. A recombinant protein, wherein said recombinant protein comprises:
(i) The antibody of any one of claims 1-6; and
(ii) Optionally a tag sequence to assist expression and/or purification.
8. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) The antibody of any one of claims 1-6; and
(2) The recombinant protein of claim 7.
9. A vector comprising the polynucleotide of claim 8.
10. A genetically engineered host cell comprising the vector or genome of claim 9 having incorporated therein the polynucleotide of claim 8.
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CN202010324347.7A Pending CN113527487A (en) | 2020-04-22 | 2020-04-22 | Monoclonal antibody of anti-human B7-H3 and application thereof |
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CN115491358B (en) * | 2021-06-17 | 2024-09-06 | 复星凯特生物科技有限公司 | Preparation and application of targeting B7-H3 and FOLR1 double-targeting CAR T |
CN116178553B (en) * | 2022-02-25 | 2023-11-17 | 南京蓬勃生物科技有限公司 | Antibodies against human B7-H3 and variants thereof |
AU2022446336A1 (en) * | 2022-03-18 | 2024-09-19 | Beijing Mabworks Biotech Co., Ltd | B7-h3-binding antibody and use thereof |
CN116396388A (en) * | 2022-08-26 | 2023-07-07 | 四川大学 | anti-B7-H3 antibody and application thereof |
TW202421666A (en) * | 2022-11-29 | 2024-06-01 | 大陸商四川科倫博泰生物醫藥股份有限公司 | B7-H3 binding protein and use thereof |
CN118515764A (en) * | 2023-01-12 | 2024-08-20 | 迈博斯(香港)科技有限公司 | Binding proteins for immunoregulatory protein molecules B7-H3 and uses thereof |
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CN106279416B (en) * | 2010-03-04 | 2019-08-30 | 宏观基因有限公司 | With the reactive antibody of B7-H3, its immunologic competence segment and application thereof |
RU2668170C2 (en) * | 2011-04-25 | 2018-09-26 | Дайити Санкио Компани, Лимитед | Anti-b7-h3-antibody |
WO2014160627A1 (en) * | 2013-03-25 | 2014-10-02 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Anti-cd276 polypeptides, proteins, and chimeric antigen receptors |
US10865245B2 (en) * | 2014-12-23 | 2020-12-15 | Full Spectrum Genetics, Inc. | Anti-B7H3 binding compounds and uses thereof |
EP3442574A4 (en) * | 2016-04-15 | 2019-12-11 | MacroGenics, Inc. | Novel b7-h3 binding molecules, antibody drug conjugates thereof and methods of use thereof |
JP6751165B2 (en) * | 2016-06-08 | 2020-09-02 | アッヴィ・インコーポレイテッド | Anti-B7-H3 antibody and antibody drug conjugate |
BR112018075649A2 (en) * | 2016-06-08 | 2019-04-09 | Abbvie Inc. | anti-b7-h3 antibodies and antibody drug conjugates |
WO2018161872A1 (en) * | 2017-03-06 | 2018-09-13 | 江苏恒瑞医药股份有限公司 | Anti-b7-h3 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof |
JP2020520382A (en) * | 2017-05-12 | 2020-07-09 | メモリアル スローン ケタリング キャンサー センター | Use of anti-B7H3 antibodies to treat cancer in the central nervous system |
WO2019225787A1 (en) * | 2018-05-24 | 2019-11-28 | 에이비엘바이오 주식회사 | Anti-b7-h3 antibody and use thereof |
CN110950953B (en) * | 2018-09-26 | 2022-05-13 | 福州拓新天成生物科技有限公司 | Monoclonal antibody against B7-H3 and application thereof in cell therapy |
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