CN110885377A - anti-CD 47/VEGF bispecific antibody and application thereof - Google Patents

anti-CD 47/VEGF bispecific antibody and application thereof Download PDF

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CN110885377A
CN110885377A CN201811058600.8A CN201811058600A CN110885377A CN 110885377 A CN110885377 A CN 110885377A CN 201811058600 A CN201811058600 A CN 201811058600A CN 110885377 A CN110885377 A CN 110885377A
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万亚坤
朱敏
沈晓宁
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Shanghai Luoqi Biomedical Technology Co ltd
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Abstract

The invention provides an anti-CD 47/VEGF bispecific antibody and application thereof, wherein the anti-CD 47/VEGF bispecific antibody comprises: (a) an anti-VEGF antibody, bevacizumab; and (b) a monovalent form of a single domain antibody against CD47 linked to the anti-VEGF antibody. The bifunctional antibody disclosed by the invention can specifically target CD47 and VEGF, effectively block the interaction between a target and a ligand thereof, effectively inhibit the proliferation of tumor cells, and retain the anti-tumor functions of a CD47 single-domain antibody and bevacizumab, and the double antibody has a good application prospect.

Description

anti-CD 47/VEGF bispecific antibody and application thereof
Technical Field
The invention belongs to the field of tumor immunology, and particularly relates to an anti-CD 47/VEGF bispecific antibody and an application thereof.
Background
Vascular epidermal growth factor family members-A (VEGF), B, C, D, and placental growth factor all belong to the cysteine knot growth factor superfamily proteins, and play important roles in angiogenesis, revascularization, and lymphangiogenesis under normal conditions (e.g., development, injury repair) and pathological conditions (e.g., tumor growth and intraocular neovascularization). VEGFA plays an important role in functions such as endothelial cell proliferation and survival, increased vascular permeability, as well as chemotaxis and homing of bone marrow hematopoietic cells, which are important for angiogenesis. VEGFA exists in multiple subtypes: 121(VEGFA), 165, 189 and 206, wherein VEGFA165 is the most commonly expressed subtype in normal and tumor tissues and is considered to be the subtype that best reflects VEGFA physiological functions. Among the receptors that bind VEGFA are VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR) and NP1/NP 2. VEGFR2 is a receptor that primarily regulates VEGFA functions related to tumor growth, such as cell proliferation, angiogenesis, and vascular permeability. Binding of VEGFA to VEGFR2 results in the formation and autophosphorylation of VEGFR2 dimer, thereby activating multiple downstream signaling pathways. VEGFR1 has a higher affinity for VEGFA than VEGFR2, but lacks a sustained mitogenic effect following VEGFR1 activation, suggesting that VEGFR1 may in some cases act as a trap receptor, modulating the action of VEGFR 2; an increasing body of data suggests that VEGFR1 plays an important role in hematopoiesis, recruitment of monocytes and other bone marrow-derived cells to home to tumor vessels, induction of matrix metalloproteinases and paracrine outgrowth of endothelial cells. AVASTIN was the first monoclonal antibody drug approved by the FDA to antagonize VEGFA for the treatment of metastatic colorectal cancer, and subsequently approved in turn for the treatment of non-small cell lung cancer, renal cell carcinoma, recurrent brain glioma, cervical cancer, metastatic breast cancer and ovarian cancer.
Bone marrow-derived cells are the most abundant immune cells in the body, however, immunotherapeutic approaches by stimulating these cells to attack cancer cells are rare. Macrophages have a strong phagocytic capacity and thus have significant potential in anti-tumor immunotherapy. However, the relationship between macrophages and tumors is complex, and in many cases, macrophages are capable of promoting the growth and spread of tumors.
CD47 is a widely expressed immunomodulatory protein, and normal cells can be protected from the killing of the immune system by binding with SIRPa on the surface of phagocyte through CD47 on the surface. The damaged cells, the aged cells have reduced expression of CD47 on their surface and are in time cleared by the immune system. Many types of tumors highly express CD47, disturbing the original CD47/SIRPa balance of the body, resulting in immune escape. The interaction of CD47/SIRPa can inhibit the killing of tumors by macrophage nucleus and other bone marrow-derived cells, and thus can be used as a bone marrow-specific immune checkpoint inhibitor.
Although VEGFA inhibitors show some efficacy in the treatment of advanced refractory malignancies and are used as standard treatment for several metastatic tumors. However, its clinical effects are relatively heterogeneous. Recent studies indicate that tumor microenvironment may modulate resistance to VEGFA inhibitors in a VEGFA independent manner.
Most of the currently marketed antibody drugs are monoclonal antibodies, and therapeutic monoclonal antibodies have been used to treat cancer, autoimmune diseases, inflammation and other diseases, most of which are specific for one target. However, patients receiving monoclonal antibody therapy may develop resistance or be unresponsive. And the factors affecting some diseases in vivo are manifold, including different signaling pathways, different cytokines and receptor regulation mechanisms, etc., and single-target immunotherapy does not seem to be sufficient to destroy cancer cells. Therefore, there is a need for a multi-targeting strategy by combining different drugs or using multispecific antibodies.
However, the development of double antibodies has been technically limited, and the greatest bottleneck is the stability of the molecular structure. The double antibody has two heavy chains and two light chains, and is very easy to generate mismatching in the development process. Moreover, the problem of segment mismatch is solved, and the molecular pharmaceutical property and large-scale production capacity are also met. These three factors are better than three supporting points of the tripod, and the industrialization of the product can be realized only by meeting all conditions. However, many technologies can only solve one or two problems, which makes the product development of bispecific antibodies always face the technical bottleneck.
Therefore, there is an urgent need in the art to develop an antitumor double antibody which has stable structure, good specificity and easy preparation, and can inhibit angiogenesis in tumors and simultaneously convert macrophages and other marrow-derived phagocytic cells in tumors from tumor assistance to tumor killing.
Disclosure of Invention
The invention aims to provide an anti-tumor double antibody which has stable structure, good specificity and easy preparation, can inhibit angiogenesis in tumors, and can convert macrophages in the tumors and other phagocytic cells derived from bone marrow from the tumor help into tumor killers.
In a first aspect, the present invention provides a bifunctional antibody comprising:
(a) an anti-VEGF antibody; and
(b) a monovalent form of a single domain antibody against CD47 linked to the anti-VEGF antibody.
In another preferred embodiment, said anti-VEGF antibody and said anti-CD 47 single domain antibody are linked by a linker peptide; preferably, the linker peptide comprises an antibody constant region sequence.
In another preferred embodiment, said single domain anti-CD 47 antibody is linked to a region of said anti-VEGF antibody selected from the group consisting of: a heavy chain variable region, a heavy chain constant region, or a combination thereof.
In another preferred embodiment, said anti-CD 47 single domain antibody is linked to the beginning of the heavy chain variable region of said anti-VEGF antibody.
In another preferred embodiment, said anti-CD 47 single domain antibody is linked to the end of the heavy chain constant region of said anti-VEGF antibody.
In another preferred embodiment, the anti-CD 47 single domain antibody comprises a humanized single domain antibody against CD 47.
In another preferred embodiment, the anti-CD 47 single domain antibody is a camel-derived single domain antibody.
In another preferred embodiment, the number of said single domain antibodies against CD47 is 1-6, preferably 2-4.
In another preferred embodiment, the bifunctional antibody is a homodimer.
In another preferred embodiment, the bifunctional antibody has a structure represented by formula I from N-terminus to C-terminus:
Figure BDA0001796473770000031
wherein the content of the first and second substances,
each D is independently a single domain antibody to CD47 or none, and at least one D is a single domain antibody to CD 47;
l1, L2 are each independently a bond or linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
In another preferred example, the Complementarity Determining Regions (CDRs) of the single domain antibody are composed of the CDR1 shown in SEQ ID No. 1, the CDR2 shown in SEQ ID No. 2 and the CDR3 shown in SEQ ID No. 3.
In another preferred embodiment, the CDRs 1, 2 and 3 of the single domain antibody are separated by the framework regions FR1, FR2, FR3 and FR 4.
In another preferred example, the FR1, FR2, FR3 and FR4 are respectively shown in SEQ ID NO. 4, 5, 6 and 7.
In another preferred embodiment, the tab members may be identical or different.
In another preferred example, the L1, L2 are each independently selected from GS, GGGGS (SEQ ID No.:8), GGGGSGGGS (SEQ ID No.:9), ggggsggggsggsggggs (SEQ ID No.: 10).
In another preferred example, the L chain (VL-CL) of the anti-VEGF antibody has the amino acid sequence shown in SEQ ID No. 11.
In another preferred example, the coding sequence of the L chain (VL-CL) of the anti-VEGF antibody has an amino acid sequence as set forth in SEQ ID No.:15, or a sequence shown in figure 15.
In another preferred example, the H chain (VH-CH) of the anti-VEGF antibody has the amino acid sequence shown in SEQ ID No. 12.
In another preferred example, the coding sequence of the H chain (VH-CH) of the anti-VEGF antibody has an amino acid sequence as set forth in SEQ ID No.: 16, or a sequence shown in figure 16.
In another preferred example, the anti-VEGF antibody comprises bevacizumab.
In another preferred embodiment, the L chain of the bifunctional antibody is selected from the group consisting of the sequences shown in SEQ ID No. 11.
In another preferred embodiment, the H chain of the bifunctional antibody is selected from the sequences as shown in SEQ ID No. 13 or SEQ ID No. 14.
In another preferred embodiment, the coding sequence of the L chain of the bifunctional antibody is selected from the sequences as shown in SEQ ID No. 15.
In another preferred embodiment, the coding sequence of the H chain of the bifunctional antibody is selected from the sequences as shown in SEQ ID No. 17 or SEQ ID No. 18.
In another preferred embodiment, the bifunctional antibody further comprises (preferably coupled to) a detectable label, a targeting label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the bifunctional antibody is coupled to a tumor targeting marker conjugate.
In another preferred embodiment, the bifunctional antibody of the present invention further comprises an active fragment and/or derivative of said antibody, said derivative comprising the active fragment of claim 1 and/or said derivative retaining 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of the anti-CD 20 and/or anti-CD 47 activity.
In another preferred embodiment, the derivative of the antibody has at least 85% sequence identity with an antibody of the invention.
In another preferred embodiment, the derivative of the antibody is a sequence of the antibody of the invention which has undergone deletion, insertion and/or substitution of one or more amino acids and which retains at least 85% identity.
In another preferred embodiment, the derivative of the antibody has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the antibody of the invention.
In another preferred embodiment, the substitution is a conservative substitution.
In another preferred embodiment, the bifunctional antibody has a structure represented by formula II from N-terminus to C-terminus:
Figure BDA0001796473770000051
wherein the content of the first and second substances,
d represents a single domain antibody against CD 47;
l2 represents no or a linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
In another preferred example, the Complementarity Determining Regions (CDRs) of the single domain antibody are composed of the CDR1 shown in SEQ ID No. 1, the CDR2 shown in SEQ ID No. 2 and the CDR3 shown in SEQ ID No. 3.
In another preferred embodiment, the CDRs 1, 2 and 3 of the single domain antibody are separated by the framework regions FR1, FR2, FR3 and FR 4.
In another preferred example, the FR1, FR2, FR3 and FR4 are respectively shown in SEQ ID NO. 4, 5, 6 and 7.
In a second aspect, the present invention provides an isolated polynucleotide encoding a bifunctional antibody according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide has a polynucleotide encoding the L chain of the bifunctional antibody as shown in SEQ ID No. 15.
In another preferred example, the polynucleotide has a polynucleotide as shown in SEQ ID No. 17 or SEQ ID No. 18 encoding the H chain of the bifunctional antibody.
In another preferred embodiment, the ratio of the polynucleotide encoding the L chain to the polynucleotide encoding the H chain is 3:2 or 1:1, preferably 3:2, in said polynucleotides.
In a third aspect, the present invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the vector contains all of the polynucleotides of the second aspect of the invention simultaneously.
In another preferred embodiment, the vectors each comprise a polynucleotide of the polynucleotides of the second aspect of the invention.
In another preferred embodiment, the vector is an expression vector.
In another preferred embodiment, the vector comprises a plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.
In a fourth aspect, the present invention provides a genetically engineered host cell comprising a vector or genome according to the third aspect of the invention into which a polynucleotide according to the second aspect of the invention has been integrated.
In a fifth aspect, the present invention provides a method of producing an antibody according to the first aspect of the invention, comprising the steps of:
(i) culturing the host cell of the fourth aspect of the invention under suitable conditions to obtain a mixture comprising the antibody of the first aspect of the invention;
(ii) (ii) purifying and/or separating the mixture obtained in step (i) to obtain the antibody according to the first aspect of the invention.
In another preferred example, the purification can be performed by protein a affinity column purification and separation to obtain the target antibody.
In another preferred embodiment, the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
In a sixth aspect, the present invention provides a pharmaceutical composition comprising:
(I) a bifunctional antibody according to the first aspect of the invention; and
(II) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises an antineoplastic agent.
In another preferred embodiment, the pharmaceutical composition is in unit dosage form.
In another preferred embodiment, the antineoplastic agent comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, or pembrolizumab.
In another preferred embodiment, the anti-neoplastic agent may be present in a separate package from the bifunctional antibody, or the anti-neoplastic agent may be conjugated to the bifunctional antibody.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a parenteral dosage form or a parenteral dosage form.
In another preferred embodiment, the parenteral dosage form comprises intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
In a seventh aspect, the invention provides an immunoconjugate comprising:
(a) a bifunctional antibody according to the first aspect of the invention; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.
In an eighth aspect, the present invention provides the use of the bifunctional antibody according to the first aspect of the present invention or the immunoconjugate according to the seventh aspect of the present invention for the preparation of a pharmaceutical composition for the treatment of tumors.
In another preferred embodiment, the tumor comprises a solid tumor, lymphoma and/or leukemia.
In another preferred embodiment, the solid tumor comprises a malignant tumor.
In another preferred embodiment, the tumor or solid tumor is selected from the group consisting of: ovarian cancer, colorectal cancer, malignant melanoma, lung cancer, gastric cancer, liver cancer, kidney cancer, head and neck squamous cancer, bladder cancer, pancreatic cancer, breast cancer, or a combination thereof.
The present invention also provides a method of treating a tumor comprising the steps of: administering to a subject in need thereof a safe and effective amount of an antibody according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
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FIG. 1 is a schematic structural diagram of an anti-CD 47/VEGF bispecific antibody of the present invention. As shown in the figure, the 2 bispecific antibodies designed by the present invention consist of CD47 single domain antibody and bevacizumab.
FIG. 2 is a graph showing the purity analysis of the anti-CD 47/CD20 bispecific antibody of the present invention. SEC detection results of the expression purified bispecific antibody show that the purity of the double-antibody A reaches 95.06 percent, and the purity of the double-antibody B reaches 97.67 percent.
FIG. 3 is the agglutination of human red blood cells by candidate diabody A. The results show that: diabody A does not cause agglutination of human red blood cells, whereas diabody B apparently causes agglutination of human red blood cells.
Figure 4 is the flow cytometry results of detecting bispecific antibody binding to both CD47 and VEGF targets. The results show that the bispecific antibody B of the present invention can simultaneously bind to CD47 and VEGF on the cell surface, and the binding capacity is similar to the binding activity of the control antibody, thus demonstrating that the bispecific antibody retains better target binding activity.
Figure 5 is the flow cytometry IC50 results for detection of bispecific antibody a. The IC50 of the bispecific antibody A is 2.923ug/mL, and the IC50 of the CD47 nanobody Nb1902-Fc is 1.590 ug/mL.
FIG. 6 shows the results of CCK8 testing for the proliferation-inhibiting toxicity of bispecific antibodies against HUVEC cells. The CCK8 kit detection result shows that the bispecific antibody A and the control antibody bevacizumab have similar proliferation inhibition effect on HUVEC cells and good biological activity.
FIG. 7 is candidate dual-antibody A-mediated phagocytosis of HUVEC cells by macrophages in vitro. The results show that: the double-antibody A can effectively promote phagocytosis of HUVEC cells by macrophages.
FIG. 8 is a test of the efficacy of candidate dual-anti-A in SK-OV-3 tumor model mice. The results show that: the tumor inhibition (TGI) of the candidate diabody a was 84%, and the tumor inhibition (TGI) of the control bevacizumab was 86%.
Fig. 9 is a test of the efficacy of candidate dual-antibody a in Raji tumor model mice. The results show that: the tumor inhibition rate of the control antibody Nb1902 was 86%, and the tumor inhibition rate of the diabody a was 88%.
Detailed Description
The present inventors have made extensive and intensive studies and as a result, have unexpectedly found that a bifunctional antibody comprising an anti-VEGF antibody and an anti-CD 47 single domain antibody connected in tandem, which is a homodimer and has a molecular weight of 175.154kD, is obtained. In vitro experiments prove that the bifunctional antibody can be combined with VEGF and CD47 simultaneously, so that the therapeutic effect on CD47 positive and VEGF positive tumor cells (particularly malignant tumor cells) is exerted, and therefore, the bifunctional antibody can be developed into an antitumor drug with excellent curative effect. On this basis, the present inventors have completed the present invention.
Term(s) for
Generally, an "antibody," also referred to as an "immunoglobulin," can be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ (l) and κ (k). There are five major heavy chain species (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain comprises a different sequence domain. The light chain comprises two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity for an antigen. The constant domains of the light Chain (CL) and heavy Chain (CH) confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light and one heavy chain. The specificity of an antibody depends on the structural complementarity of the antibody binding site and the epitope. The antibody binding site consists of residues derived primarily from the hypervariable region or Complementarity Determining Region (CDR). Occasionally, residues from non-highly variable or Framework Regions (FR) affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, otherwise designated as CDRs 1-L, CDR2-L, CDR3-L and CDRs 1-H, CDR2-H, CDR 3-H. Conventional antibody antigen binding sites therefore include six CDRs, comprising a collection of CDRs from each heavy and light chain v region.
As used herein, the terms "single domain antibody", "VHH", "nanobody" have the same meaning and refer to the variable region of a cloned antibody heavy chain, and a single domain antibody constructed consisting of only one heavy chain variable region is the smallest antigen-binding fragment with full function. Typically, single domain antibodies consisting of only one heavy chain variable region are constructed by first obtaining an antibody that is naturally deficient in light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
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 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 segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially b-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a b-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIHPubl. No.91-3242, Vol.I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
As used herein, the term "framework region" (FR) refers to amino acid sequences inserted between CDRs, i.e., those portions of the light and heavy chain variable regions of an immunoglobulin that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR 4-H. Accordingly, the light chain variable domain may thus be referred to as (FR1-L) - (CDR1-L) - (FR2-L) - (CDR2-L) - (FR3-L) - (CDR3-L) - (FR4-L) and the heavy chain variable domain may thus be referred to as (FR1-H) - (CDR1-H) - (FR2-H) - (CDR2-H) - (FR3-H) - (CDR3-H) - (FR 4-H). Preferably, the FRs of the present invention are human antibody FRs or derivatives thereof that are substantially identical, i.e., 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, to the FRs of a naturally occurring human antibody.
Knowing the amino acid sequences of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR 4-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework regions of a naturally occurring human antibody.
As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule having a single amino acid composition to a particular antigen, and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but can also be recombinant, i.e., produced by protein engineering.
As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule capable of being bound by an antibody or antibody-like binding protein. The term further refers to a molecule or portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. The target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein is capable of competing with the intact antibody recognizing the target antigen.
As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into one or more amino acid residues that exchange the dual variable region immunoglobulin. The linker of the invention refers to linkers L1 and L2, wherein L1 connects the heavy chain variable region VH of the anti-CD 47 single domain antibody of the double antibody of the invention and the anti-VEGF antibody, and L2 connects the heavy chain constant region CH of the anti-CD 47 single domain antibody of the double antibody of the invention and the anti-VEGF antibody.
Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element that is desired to be implemented in the linker. . One preferred joint is as follows:
l1 and L2 are each independently selected from the following sequences: GS, GGGGS (SEQ ID NO.:8), GGGGSGGGS (SEQ ID NO.:9), GGGGSGGGGSGGSGGGGS (SEQ ID NO.: 10).
anti-CD 47 antibodies
CD47 is a widely expressed immunomodulatory protein, and normal cells can be protected from the killing of the immune system by binding with SIRPa on the surface of phagocyte through CD47 on the surface. The damaged cells, the aged cells have reduced expression of CD47 on their surface and are in time cleared by the immune system. Many types of tumors highly express CD47, disturbing the original CD47/SIRPa balance of the body, resulting in immune escape. The interaction of CD47/SIRPa can inhibit the killing of tumors by macrophage nucleus and other bone marrow-derived cells, and thus can be used as a bone marrow-specific immune checkpoint inhibitor.
The sequence of the anti-CD 47 antibody of the invention is as described in patent application CN201810151752.6, and those skilled in the art can also modify or modify the anti-CD 47 antibody of the invention by techniques well known in the art, such as adding, deleting and/or substituting one or several amino acid residues, thereby further increasing the affinity or structural stability of anti-CD 47, and obtaining the modified or modified result by conventional assay methods.
Preferably, the anti-CD 47 antibodies of the invention, regardless of which end of the anti-VEGF antibody is attached, are attached to a single anti-CD 47 antibody by a linker so as to appear as a dimer. The anti-CD 47 antibody of the invention can be obtained by expression of HEK293 cells or CHO cells.
The anti-CD 47 antibodies of the invention bind to mammalian CD47, preferably human CD 47.
anti-VEGF antibodies
Vascular epidermal growth factor family members-A (VEGF), B, C, D, and placental growth factor all belong to the cysteine knot growth factor superfamily proteins, and play important roles in angiogenesis, revascularization, and lymphangiogenesis under normal conditions (e.g., development, injury repair) and pathological conditions (e.g., tumor growth and intraocular neovascularization). VEGFA plays an important role in functions such as endothelial cell proliferation and survival, increased vascular permeability, as well as chemotaxis and homing of bone marrow hematopoietic cells, which are important for angiogenesis. VEGFA exists in multiple subtypes: 121(VEGFA), 165, 189 and 206, wherein VEGFA165 is the most commonly expressed subtype in normal and tumor tissues and is considered to be the subtype that best reflects VEGFA physiological functions. Among the receptors that bind VEGFA are VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR) and NP1/NP 2. VEGFR2 is a receptor that primarily regulates VEGFA functions related to tumor growth, such as cell proliferation, angiogenesis, and vascular permeability. Binding of VEGFA to VEGFR2 results in the formation and autophosphorylation of VEGFR2 dimer, thereby activating multiple downstream signaling pathways. VEGFR1 has a higher affinity for VEGFA than VEGFR2, but lacks a sustained mitogenic effect following VEGFR1 activation, suggesting that VEGFR1 may in some cases act as a trap receptor, modulating the action of VEGFR 2; an increasing body of data suggests that VEGFR1 plays an important role in hematopoiesis, recruitment of monocytes and other bone marrow-derived cells to home to tumor vessels, induction of matrix metalloproteinases and paracrine outgrowth of endothelial cells. AVASTIN was the first monoclonal antibody drug approved by the FDA to antagonize VEGFA for the treatment of metastatic colorectal cancer, and subsequently approved in turn for the treatment of non-small cell lung cancer, renal cell carcinoma, recurrent brain glioma, cervical cancer, metastatic breast cancer and ovarian cancer.
The anti-VEGF antibody useful in the present invention consists of a light chain shown in SEQ ID No. 11 and a heavy chain shown in SEQ ID No. 12.
Bifunctional antibodies (bispecific antibodies)
As used herein, the terms "bispecific antibody," "bifunctional antibody," "antibody of the invention," "dual anti," "dual-anti," "bifunctional fusion antibody" are used interchangeably to refer to an anti-VEGF/CD 47 bispecific antibody that binds both VEGF and CD 47.
In the present invention, the bifunctional antibody comprises:
(a) an anti-VEGF antibody; and
(b) a monovalent form of a single domain antibody against CD47 linked to the anti-VEGF antibody.
In a preferred embodiment, the bifunctional antibody of the present invention has, from N-terminus to C-terminus, the structure of formula I:
Figure BDA0001796473770000121
wherein the content of the first and second substances,
each D is independently a single domain antibody to CD47, at least one D being a single domain antibody to CD 47;
l1, L2 are each independently a bond or linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
In a preferred embodiment, the double antibody of the present invention is a fusion of an anti-VEGF antibody and a single domain antibody of anti-CD 47, and has two pairs of peptide chains symmetrical to each other, each pair of peptide chains comprising a light chain L chain and a heavy chain H chain, all of which are linked by a disulfide bond, wherein any one pair of peptide chains has, from N-terminus to C-terminus, the L chain and H chain structures shown in formula II:
Figure BDA0001796473770000131
wherein the content of the first and second substances,
d represents a single domain antibody with anti-CD 47;
l2 represents no or a linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
In formula I or formula II, a preferred L chain is shown in SEQ ID No. 11, and a preferred H chain is shown in SEQ ID No. 13 or 14.
The sequence of the coding L chain is shown in SEQ ID NO. 15, and the sequence of the coding H chain is shown in SEQ ID NO. 17 or 18. (same front)
And the two sequences shown in the structural formula I are connected through a disulfide bond of an H chain, so that a symmetrical bifunctional antibody structure is formed.
The double antibodies of the invention include not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. 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 those skilled in the art in view of the teachings herein.
The double antibodies of the invention refer to antibodies with anti-CD 47 and anti-VEGF activities that include two structures of formula I above. The term also includes variants of the antibody having the same function as the diabodies of the invention, including the two structures of formula I above. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the double antibodies of the invention.
The variant forms of the double antibody include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
In the present invention, "conservative variant of the diabody of the present invention" refers to that 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 having similar or similar properties as compared to the amino acid sequence of the diabody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
Figure BDA0001796473770000141
Figure BDA0001796473770000151
Coding nucleic acids and expression vectors
The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
The nucleic acids (and combinations of nucleic acids) of the invention can be used to produce recombinant antibodies of the invention in a suitable expression system.
The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the mature polypeptide encode polypeptides having the same biological functions and activities as the mature polypeptide.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express 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. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The obtained transformant 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 culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These 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 (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
The diabodies of the present invention may be used alone, in combination or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. tumor therapeutic agents (e.g., cisplatin) or any form of antineoplastic agent, and the like.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will 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: intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.
The pharmaceutical composition of the invention can be directly used for binding CD47 protein molecules or VEGF, and thus can be used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single domain antibody (or conjugate thereof) of the present invention 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 preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 10 micrograms per kilogram of body weight to about 50 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 case of pharmaceutical compositions, 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/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention include:
1. the bifunctional antibody can simultaneously bind VEGF and CD47, and keeps the binding configuration of the bifunctional antibody and a binding target unchanged, so that the molecule is stable.
2. The bifunctional antibody can be expressed in HEK293F cells, the purity of the bifunctional antibody purified by one-step affinity chromatography can reach more than 95%, and the preparation method is simple, convenient and feasible.
3. The bifunctional antibody can effectively combine double targets of CD47 and VEGF simultaneously, and keeps better combination activity and blocking activity.
4. The bifunctional antibody of the invention does not cause human erythrocyte agglutination and shows better safety.
5. The bifunctional antibody disclosed by the invention not only has good biological activity of a CD47 antibody, but also perfectly maintains the biological activity of bevacizumab, and shows obvious anti-tumor activity in a mouse efficacy model.
Therefore, the anti-CD 47/VEGF bispecific antibody provided by the invention has a good application prospect.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The materials or reagents used in the examples are all commercially available products unless otherwise specified.
Example 1 anti-CD 47/VEGF bispecific antibody molecule sequence design
The structure of the bispecific antibody which can simultaneously bind to CD47 and VEGF extracellular domain is shown in figure 1.CD47 single domain antibody sequences were derived from patent 201810151752.6; the above CD47 single domain antibody was then fused to the heavy chain of bevacizumab, which was linked to bevacizumab heavy chain (LALA) and CD47 single domain antibody by Linker ((GGGGS)4), with the following amino acid sequences (the underlined part is the Linker sequence):
a: 13, the CD47 single-domain antibody is connected to the C end of the heavy chain of the bevacizumab through a linker (GGGGS) 4;
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSS
b: 14, wherein the CD47 single-domain antibody is connected to the N end of the heavy chain of the bevacizumab through a linker (GGGGS) 4;
QVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK the light chain of the bispecific antibody molecule is the bevacizumab light chain as shown in SEQ ID No. 11;
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
the amino acid sequences of the heavy chain and the light chain are respectively converted into base sequences, the base sequences of the heavy chain are shown as SEQ IDON 17 and SEQ ID ON.:18, and the base sequences of the light chain are shown as SEQ ID ON.: 15. The above base sequences were synthesized to expression vectors pCDNA3.1(+) respectively.
Example 2 anti-CD 47/VEGF bispecific antibody expression purification
The two plasmids in which the synthetic genes are located are transiently transferred into HEK293F cells together, and the specific method is as follows: (1) using OMEGA plasmid answering kit to respectively classify the plasmid of heavy chain and the plasmid of light chainRespectively preparing a large amount of granules, and filtering and sterilizing the granules in a superclean workbench for later use; (2) HEK293F cells were cultured to 2.0X 106Per mL; (3) heavy and light chain plasmids were made according to 2: 3, the mixed plasmid and the transfection reagent PEI are evenly mixed in a ratio of 1:3 into a transfection medium F17(Gibco), the mixture is kept stand for 20min, and then the mixture is added into HEK293F cells at 37 ℃ and 6% CO2Culturing for 6 days in a shaking incubator; (4) centrifuging to obtain a supernatant, and combining the supernatant with protein A beads for 1h at room temperature; (5) washing away the hybrid protein and other impurities by using phosphate buffer solution with pH7.0, then eluting the target antibody protein by using Glycine with 0.1MpH3.0, and numbering the obtained anti-CD 47/VEGF antibody as double-anti-A and double-anti-B; (6) ultrafiltering the eluted antibody into 1 × PBS solution, subpackaging and storing at-80 deg.C.
TABLE 1 CD47/VEGF bispecific antibody sequence composition
Figure BDA0001796473770000201
Example 3 purity assay of candidate diabody A
The purified sample, double-antibody A, was subjected to SEC analysis: (1) sample preparation: the sample was diluted to 1.0mg/mL with the mobile phase as a test sample. The sample was filtered through a 0.22um needle filter into a sample vial and placed in an HPLC autosampler to prepare for injection. (2) Mobile phase: 200mM phosphate in water, pH7.0, column: x bridge BEH200SEC3.5 μm 7.8X300mm, parameters set as follows: the detection wavelength is 280nm, the column temperature is 25 ℃, the flow rate is 0.5mL/min, and the sample injection amount is 10 uL. (3) The detection result is shown in fig. 2, the purity of the diabody A reaches 95.06%, and the purity of the diabody B reaches 97.67%, and the diabody B can be used for subsequent experiments.
Example 4 determination of agglutination of human Red blood cells by candidate diabodies
Because the surface of the erythrocyte has high expression of CD47, the erythrocyte can be more easily and preferentially combined with CD47 antibody drugs, and the drugs are concentrated on the surface to play a role of a water storage tank. Therefore, anemia is likely to occur in this case. Only after the medicine enters the body, the medicine firstly needs to break through the 'absorption pool' effect of the platelets on the CD47 antibody, and can effectively reach the action position to play a role. The positive control antibody (B6H12), the CD47 single domain antibody Nb1902 (derived from patent 201810151752.6), and the negative control (IgG4) were each diluted in gradient (final concentrations of action 4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 3.906nM, 0.976nM, 0 nM). The antibodies diluted in the gradient were mixed with an equal volume of human erythrocyte suspension (2%) and left to react overnight at 37 ℃ before observing the results. The results show (FIG. 3) that candidate diabody B caused agglutination of erythrocytes more clearly, whereas candidate diabody A did not cause agglutination of erythrocytes. The results of the control antibody (B6H12) were consistent with those reported by Penkas. Petrova et al, 2016, Cl in. cancer. Res.23 (4).
Example 5FACS detection of antibodies binding to both CD47 and VEGF
The specific process is as follows: (1) taking 3X10 samples of each5Adding diluted anti-CD 47/VEGF bispecific antibody and a control antibody (the final concentration gradient of antibody dilution is 20ug/mL) into a PBS buffer, and incubating at 4 ℃ for 20 min; (2) FITC Goat anti Human IgG staining, 4 degrees C were incubated for 20 min; washing with PBS for 2 times; (3) adding VEGF165A-biotin20ug/ml, and incubating at 4 ℃ for 20 min; washing with PBS for 2 times; (4) 100uL of SA-PE staining solution was pipetted into each well using a row gun, resuspended, incubated at 4 ℃ for 20min, washed 2 times with PBS and detected using a flow cytometer (BD FACS Cal ibur), and data processed using graphpadprist 6 software. The results are shown in fig. 4, and the diabody a can bind to both CD47 and VEGF.
Example 6FACS detection of IC50 of antibodies
The specific process is as follows: (1) taking 3X10 samples of each5293T/hCD47 cells were stably transfected into PBS buffer, and a gradient of anti-CD 47/VEGF bispecific and control antibodies (final concentration gradient of antibody dilution 40ug/mL, 20ug/mL, 10ug/mL, 5ug/mL, 3.33g/mL, 2.5ug/mL, 1.67ug/mL, 1.25ug/mL, 0.84ug/mL, 0.63ug/mL, 0.42ug/mL, 0.32ug/mL) were added, 50uL of each sample, 120ug/mL hSIRPa (ECD) -Fc-Biotin 50uL were added to all samples at the same time, and incubation was carried out at 4 ℃ for 20 min; (2) washing the cells with PBS for 2 times, adding SA-PE from eBioscience, incubating at 4 deg.C for 20min, washing the cells with PBS for 2 times, detecting with flow cytometer (BD FACS Calibur), and using graph pand the adprimm 6 software performs data processing. The results are shown in FIG. 5, IC50 for bispecific antibody A is 2.923ug/mL, while IC50 for the control antibody (Nb1902-Fc) is 1.590 ug/mL.
Example 7CCK8 detection of the proliferation inhibition of HUVEC cells by Diantibody A
Specifically, (1) two 96-well plates A and B are taken, wherein the plate A is used for inoculating cells, and the plate B is used for diluting antibodies. (2) HUVEC cells were trypsinized, medium neutralized, washed once with PBS, 3X104Resuspended at a concentration of/ml (DMEM + 2% FBS) and pipetted at 100 uL/well into 96-well plates at 37 ℃ with 5% CO2 for 12. + -. 2 h. (3) The antibody (DMEM with 2% FBS) was diluted 1h before 24h of cell culture at a concentration gradient of 20000ng/ml, 5000ng/ml, 1000ng/ml, 800ng/ml, 600ng/ml, 400ng/ml, 200ng/ml, 100ng/ml, 20 ng/ml. (4) The medium in the cells was aspirated and 100uL of diluted antibody was added. (5) After 72h, adding 10 uL/well of CCK8 solution, developing for 1-4h, and reading OD450 of each well by using a microplate reader after the color development is finished. The experimental results shown in fig. 6 indicate that the inhibitory activity of the anti-CD 47/VEGF bispecific antibody of the present invention on HUVEC cell proliferation is the same as that of bevacizumab, which indicates that the bispecific antibody of the present invention better retains the biological activity of bevacizumab.
Example 8 candidate diabody A mediates in vitro macrophage phagocytosis of Raji cells
The specific process is as follows: (1) PBMCs were induced in RPMI 1640 (10% FBS) containing 40ng/ml M-CSF for 9-12 days and macrophages were harvested. (2) Raji was labeled with CFSE (final concentration: 1.5uM), and macrophage was labeled with eFlour670 (final concentration: 1.25 uM). (3) The monoclonal antibody was prepared by mixing macrohage 1E5(100uL) and 3E5(50uL) Raji, and adding 50uL of the diluted antibody. (4)37 ℃ and 5% CO2 for 3 h. (5) The cells in each well were transferred to a 1.5ml EP tube, after which each well was washed with 200uL of PBS, and then the washed PBS was transferred to the corresponding 1.5ml EP tube. (6) Centrifuge at 3000rpm for 4min at 4 ℃ taking care not to aspirate the cells, add 500uL PBS/tube, and resuspend. (7) Centrifuge at 3000rpm at 4 ℃ for 4min, add 200uL PBS/well, resuspend, flow assay. The results are shown in FIG. 7: the candidate double-antibody A can effectively promote phagocytosis of HUVEC cells by macrophages.
Example 9 detection of drug efficacy of candidate diabodies A in SK-OV-3 tumor model mice
Will contain 5x 106A0.1 mL (DPBS) cell suspension of SK-OV-3 cells and 0.1mL matri-gel were mixed well and inoculated subcutaneously in the right back of each mouse (BALB/c nude, 4-6 weeks). The average tumor volume reaches about 50-100mm3The grouped administration is started. Dose of antibody: 5 mg/kg. The administration was 2 times per week for 4 weeks. The results in FIG. 8 show that: the tumor inhibition ratio (TGI) of the experimental group of the dual anti-a was 84%, and the tumor inhibition ratio (TGI) of the control bevacizumab was 86%. Therefore, the tumor inhibition effect of the candidate double-antibody A and bevacizumab in mice is similar.
Example 10 detection of drug efficacy of candidate diabodies A in Raji tumor model mice
Will contain 5X 106A 0.2ml cell suspension of Raji cells (cells suspended in DPBS) was inoculated subcutaneously into the right hind-dorsal (NOG mice, 4-6 weeks) of each mouse. The average tumor volume reaches about 50-100mm3The grouped administration is started. Dose of antibody: 20 mg/kg. Daily dosing was continued for 4 weeks. The results in FIG. 9 show that: in the model, the tumor inhibition rate of the double-anti-A is 88%, the tumor inhibition rate of the control antibody Nb1902 is 86%, and the candidate double-anti-A also well retains the anti-tumor effect of the CD47 antibody.
The above examples show that the anti-CD 47/VEGF bispecific antibody of the present invention can be expressed in HEK293F cells, can be further purified by affinity chromatography, and can reach a purity of 99.33% after only one-step purification. The resulting bispecific antibody can simultaneously bind to CD47 positive Raji cells and VEGF expressing SK-OV-3 cells and simultaneously block the interaction between cell surface antigens and their ligands. The antibody can effectively promote the phagocytosis of macrophages on tumor cells, simultaneously play the role of bevacizumab in inhibiting the proliferation of tumor cells, and cannot cause human erythrocyte agglutination; in addition, the antibody can exert double-target anti-tumor effect in mice. Therefore, the anti-CD 47/VEGF bispecific antibody provided by the invention has a good application prospect.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Luoqi biomedical technology, Inc
<120> anti-CD 47/VEGF bispecific antibody and application thereof
<130>P2018-1175
<160>18
<170>PatentIn version 3.5
<210>1
<211>10
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>1
Gly Tyr Ala Tyr Thr Ser Asp Cys Met Gly
1 5 10
<210>2
<211>8
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>2
Ile Tyr Thr Pro Gly Asn Ser Thr
1 5
<210>3
<211>15
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>3
Ala Ala Arg Arg Gly Ala Cys Ser Leu Arg Leu Pro Phe Phe Tyr
1 5 10 15
<210>4
<211>25
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>4
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210>5
<211>15
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>5
Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val Ala Leu
1 5 10 15
<210>6
<211>38
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>6
Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210>7
<211>11
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>7
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210>8
<211>5
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>8
Gly Gly Gly Gly Ser
1 5
<210>9
<211>9
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>9
Gly Gly Gly Gly Ser Gly Gly Gly Ser
1 5
<210>10
<211>20
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>10
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210>11
<211>214
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His 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 Tyr Ser Thr Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210>12
<211>453
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
225 230 235 240
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210>13
<211>595
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
225 230 235 240
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
450 455 460
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser
465 470 475 480
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
485 490 495
Ala Ser Gly Tyr Ala Tyr Thr Ser Asp Cys Met Gly Trp Phe Arg Gln
500 505 510
Thr Pro Gly Lys Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly
515 520 525
Asn Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
530 535 540
Gln Asp Asn Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg
545 550 555 560
Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys
565 570 575
Ser Leu Arg Leu Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr
580 585 590
Val Ser Ser
595
<210>14
<211>595
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>14
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Thr Ser Asp
20 25 30
Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Leu Ile Tyr Thr Pro Gly Asn Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Arg Arg Gly Ala Cys Ser Leu Arg Leu Pro Phe Phe Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
130 135 140
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
145 150 155 160
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met
165 170 175
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp
180 185 190
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg
195 200 205
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln
210 215 220
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys
225 230 235 240
Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly
245 250 255
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
260 265 270
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
275 280 285
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
290 295 300
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
305 310 315 320
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
325 330 335
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
340 345 350
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
355 360 365
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
370 375 380
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
385 390 395 400
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
405 410 415
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
420 425 430
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
435 440 445
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
450 455 460
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
465 470 475 480
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
485 490 495
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
500 505 510
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
515 520 525
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
530 535 540
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
545 550 555 560
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
565 570 575
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
580 585 590
Pro Gly Lys
595
<210>15
<211>642
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>15
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60
atcacctgca gcgccagcca ggacatcagc aactacctga actggtacca gcagaagccc 120
ggcaaggccc ccaaggtgct gatctacttc accagcagcc tgcacagcgg cgtgcccagc 180
aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccctggac cttcggccag 300
ggcaccaagg tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480
gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642
<210>16
<211>1359
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>16
gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta caccttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac 180
gccgccgact tcaagaggag gttcaccttc agcctggaca ccagcaagag caccgcctac 240
ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caagtacccc 300
cactactacg gcagcagcca ctggtacttc gacgtgtggg gccagggcac cctggtgacc 360
gtgagcagcg ccagcaccaa gggccccagc gtgttccccc tggcccccag cagcaagagc 420
accagcggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 480
accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg 540
cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc 600
acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaagaag 660
gtggagccca agagctgcga caagacccac acctgccccc cctgccccgc ccccgaggcc 720
gccggcggcc ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 780
aggacccccg aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag 840
ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 900
cagtacaaca gcacctacag ggtggtgagc gtgctgaccg tgctgcacca ggactggctg 960
aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcccgcccc catcgagaag 1020
accatcagca aggccaaggg ccagcccagg gagccccagg tgtacaccct gccccccagc 1080
agggaggaga tgaccaagaa ccaggtgagc ctgacctgcc tggtgaaggg cttctacccc 1140
agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc 1200
ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 1260
agcaggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1320
cactacaccc agaagagcct gagcctgagc cccggcaag 1359
<210>17
<211>1785
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>17
gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta caccttcacc aactacggca tgaactgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac 180
gccgccgact tcaagaggag gttcaccttc agcctggaca ccagcaagag caccgcctac 240
ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caagtacccc 300
cactactacg gcagcagcca ctggtacttc gacgtgtggg gccagggcac cctggtgacc 360
gtgagcagcg ccagcaccaa gggccccagc gtgttccccc tggcccccag cagcaagagc 420
accagcggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 480
accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg 540
cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc 600
acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaagaag 660
gtggagccca agagctgcga caagacccac acctgccccc cctgccccgc ccccgaggcc 720
gccggcggcc ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 780
aggacccccg aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag 840
ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 900
cagtacaaca gcacctacag ggtggtgagc gtgctgaccg tgctgcacca ggactggctg 960
aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcccgcccc catcgagaag 1020
accatcagca aggccaaggg ccagcccagg gagccccagg tgtacaccct gccccccagc 1080
agggaggaga tgaccaagaa ccaggtgagc ctgacctgcc tggtgaaggg cttctacccc 1140
agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc 1200
ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 1260
agcaggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1320
cactacaccc agaagagcct gagcctgagc cccggcaagg gcggcggcgg cagcggcggc 1380
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcc aggtgcagct gcaggagtcc 1440
ggcggcggcc tggtgcagcc cggcggctcc ctgaggctgt cctgcgccgc ctccggctac 1500
gcctacacct ccgactgcat gggctggttc aggcagaccc ccggcaaggg cctggagggc 1560
gtggccctga tctacacccc cggcaactcc accaactacg ccgactccgt gaagggcagg 1620
ttcaccatct cccaggacaa ctccaagtcc accgtgtacc tgcagatgaa ctccctgagg 1680
gccgaggaca ccgccatgta ctactgcgcc gccaggaggg gcgcctgctc cctgaggctg 1740
cccttcttct actggggcca gggcaccctg gtgaccgtgt cctcc 1785
<210>18
<211>1785
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>18
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctacacc tccgactgca tgggctggtt caggcagacc 120
cccggcaagg gcctggaggg cgtggccctg atctacaccc ccggcaactc caccaactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca actccaagtc caccgtgtac 240
ctgcagatga actccctgag ggccgaggac accgccatgt actactgcgc cgccaggagg 300
ggcgcctgct ccctgaggct gcccttcttc tactggggcc agggcaccct ggtgaccgtg 360
tcctccggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 420
ggcagcgagg tgcagctggt ggagagcggc ggcggcctgg tgcagcccgg cggcagcctg 480
aggctgagct gcgccgccag cggctacacc ttcaccaact acggcatgaa ctgggtgagg 540
caggcccccg gcaagggcct ggagtgggtg ggctggatca acacctacac cggcgagccc 600
acctacgccg ccgacttcaa gaggaggttc accttcagcc tggacaccag caagagcacc 660
gcctacctgc agatgaacag cctgagggcc gaggacaccg ccgtgtacta ctgcgccaag 720
tacccccact actacggcag cagccactgg tacttcgacg tgtggggcca gggcaccctg 780
gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc ccccagcagc 840
aagagcacca gcggcggcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag 900
cccgtgaccg tgagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc 960
gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc 1020
ctgggcaccc agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaggtggac 1080
aagaaggtgg agcccaagag ctgcgacaag acccacacct gccccccctg ccccgccccc 1140
gaggccgccg gcggccccag cgtgttcctg ttccccccca agcccaagga caccctgatg 1200
atcagcagga cccccgaggt gacctgcgtg gtggtggacg tgagccacga ggaccccgag 1260
gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccagg 1320
gaggagcagt acaacagcac ctacagggtg gtgagcgtgc tgaccgtgct gcaccaggac 1380
tggctgaacg gcaaggagta caagtgcaag gtgagcaaca aggccctgcc cgcccccatc 1440
gagaagacca tcagcaaggc caagggccag cccagggagc cccaggtgta caccctgccc 1500
cccagcaggg aggagatgac caagaaccag gtgagcctga cctgcctggt gaagggcttc 1560
taccccagcg acatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag 1620
accacccccc ccgtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg 1680
gacaagagca ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg 1740
cacaaccact acacccagaa gagcctgagc ctgagccccg gcaag 1785

Claims (10)

1. A bifunctional antibody, wherein said bifunctional antibody comprises:
(a) an anti-VEGF antibody; and
(b) a monovalent form of a single domain antibody against CD47 linked to the anti-VEGF antibody.
2. The bifunctional antibody of claim 1, wherein the bifunctional antibody has a structure represented by formula I from N-terminus to C-terminus:
Figure FDA0001796473760000011
wherein the content of the first and second substances,
each D is independently a single domain antibody to CD47 or none, and at least one D is a single domain antibody to CD 47;
l1, L2 are each independently a bond or linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
3. The bifunctional antibody of claim 1, wherein the bifunctional antibody has a structure represented by formula II from N-terminus to C-terminus:
Figure FDA0001796473760000012
wherein the content of the first and second substances,
d represents a single domain antibody against CD 47;
l2 represents no or a linker element;
VL represents the light chain variable region of an anti-VEGF antibody;
CL represents the light chain constant region of an anti-VEGF antibody;
VH represents the heavy chain variable region of an anti-VEGF antibody;
CH represents the heavy chain constant region of an anti-VEGF antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding VEGF and CD 47.
4. An isolated polynucleotide encoding the bifunctional antibody of claim 1.
5. A vector comprising the polynucleotide of claim 4.
6. A genetically engineered host cell comprising the vector of claim 5 or having the polynucleotide of claim 4 integrated into its genome.
7. A method of producing the antibody of claim 1, comprising the steps of:
(i) culturing the host cell of claim 6 under suitable conditions to obtain a mixture comprising the antibody of claim 1;
(ii) (ii) purifying and/or separating the mixture obtained in step (i) to obtain the antibody of claim 1.
8. A pharmaceutical composition, comprising:
(I) the bifunctional antibody of claim 1; and
(II) a pharmaceutically acceptable carrier.
9. An immunoconjugate, wherein the immunoconjugate comprises:
(a) the bifunctional antibody of claim 1; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
10. Use of a bifunctional antibody as defined in claim 1 or an immunoconjugate as defined in claim 9 for the preparation of a pharmaceutical composition for the treatment of tumors.
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