CN110885376A - anti-CD 47/CD20 bispecific antibodies and uses thereof - Google Patents

anti-CD 47/CD20 bispecific antibodies and uses thereof Download PDF

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CN110885376A
CN110885376A CN201811057987.5A CN201811057987A CN110885376A CN 110885376 A CN110885376 A CN 110885376A CN 201811057987 A CN201811057987 A CN 201811057987A CN 110885376 A CN110885376 A CN 110885376A
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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 present invention relates to anti-CD 47/CD20 bispecific antibodies and uses thereof. In particular, the invention provides anti-CD 47/CD20 bispecific antibodies comprising: (a) antibodies against CD20 (e.g., rituximab); and (b) a monovalent form of a nanobody against CD47 linked to the anti-CD 20 antibody. The bifunctional antibody has good stability, can specifically target CD47 and CD20, effectively block the interaction between CD47 and a ligand SIRPa thereof, effectively combine with cell surface CD20, and obviously inhibit tumor cell proliferation, thereby realizing the synergistic effect of the bifunctional antibody and having good application prospect.

Description

anti-CD 47/CD20 bispecific antibodies and uses thereof
Technical Field
The invention belongs to the field of tumor immunology, and particularly relates to an anti-CD 47/CD20 bispecific antibody and application thereof.
Background
The CD47 molecule, which is a highly glycosylated transmembrane protein of about 50kD, consists of an extracellular N-terminal immunoglobulin variable domain and 5 transmembrane segments with highly hydrophobic extensions and 1 short, selectively spliced cytoplasmic domain CD47 is widely distributed and expressed on the surface of myeloid cells such as macrophages, granulocytes, dendritic cells, mast cells and hematopoietic stem cells, and is highly expressed particularly in hematopoietic stem cells CD47 is a pluripotent molecule whose major function includes binding ① to CD47 ligand signaling regulatory protein (SIRP) α and interacting with Integrin, modulating intercellular communication between CD47 and SIRP α on the surface of macrophages, activating tyrosine phosphorylase, inhibiting the accumulation of myosin under the synaptic membrane, as an "separate" marker ", which interacts with the extracellular signaling protein, and is involved in the proliferation of various types of myeloid tumors, such as acute myeloid leukemia, chronic myeloproliferative leukemia, acute myeloproliferative leukemia, chronic myeloproliferative leukemia, acute myeloproliferative leukemia, chronic myeloproliferative leukemia, leukemia.
Although this approach was shown to have some effect in inducing macrophage phagocytosis in approaches that block the CD47-SIRPa signaling pathway using blocking antibodies to CD47 or SIRPa fusion proteins, such a strategy does not meet the need for tumor targeting. CD47 expressed by normal tissues produces an "antigen precipitate" of the therapeutic CD47 antibody, which in turn affects the therapeutic CD47 antibody to the tumor site in the body.
The transmembrane protein on the B lymphocyte of the leukocyte differentiation antigen 20(CD20) exists in a non-glycosylated form, is a phosphorylated protein molecule with the molecular weight of 33-37KD, is positioned on the surface of the B lymphocyte, and is the differentiation antigen on the surface of the B lymphocyte. Because of its expression in tumor cells of B cell-derived lymphomas, leukemias, and the like, and B cells involved in immune and inflammatory diseases, it has become a target for the treatment of lymphomas, leukemias, and certain autoimmune diseases.
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 anti-tumor double antibody that is structurally stable, specific, easy to prepare, and can reduce the antigen precipitating effect of normal tissues on therapeutic CD47 antibodies.
Disclosure of Invention
The invention aims to provide an anti-tumor double antibody which has stable structure, good specificity and easy preparation and can reduce the antigen precipitation effect of normal tissues on a therapeutic CD47 antibody.
In a first aspect, the present invention provides a bifunctional antibody comprising:
(a) antibodies against CD 20; and
(b) a monovalent form of a nanobody against CD47 linked to the anti-CD 20 antibody.
In another preferred embodiment, the anti-CD 20 antibody and the anti-CD 47 nanobody are linked by a linker peptide; preferably, the linker peptide comprises an antibody constant region sequence.
In another preferred example, the nanobody of anti-CD 47 is linked to a region of the antibody of anti-CD 20 selected from the group consisting of: a heavy chain variable region, a heavy chain constant region, a light chain variable region, or a combination thereof.
In another preferred example, the nanobody of anti-CD 47 is linked to the beginning of the heavy chain variable region, and/or the light chain variable region of the anti-CD 20 antibody.
In another preferred example, the nanobody of anti-CD 47 is linked to the end of the heavy chain constant region of the antibody of anti-CD 20.
In another preferred embodiment, the anti-CD 47 nanobody comprises a humanized nanobody of anti-CD 47.
In another preferred embodiment, the number of the nanobodies against CD47 is 1-6, preferably 2-6.
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 BDA0001796306540000031
wherein the content of the first and second substances,
each D is independently a nanobody that is absent or anti-CD 47, and at least one D is a nanobody that is anti-CD 47;
l1, L2, L3 are each independently a key or linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to CD 47.
In another preferred example, the Complementarity Determining Regions (CDRs) of the nanobody consist 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 nanobody are separated by 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, L3 are each independently selected from GS, GGGGS (SEQ ID No.:8), GGGGSGGGS (SEQ ID No.:9), ggggsggggsggsggggs (SEQ ID No.: 10).
In another preferred embodiment, the anti-CD 20 antibody comprises rituximab.
In another preferred example, the L chain of the anti-CD 20 antibody has the amino acid sequence shown in SEQ ID No. 11.
In another preferred example, the H chain of the anti-CD 20 antibody has the amino acid sequence shown in SEQ ID No. 12.
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 or SEQ ID No. 13.
In another preferred embodiment, the H chain of the bifunctional antibody is selected from the group consisting of the sequences as shown in SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16.
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. 17 or SEQ ID No. 19.
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. 20 or SEQ ID No. 21 or SEQ ID No. 22.
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 BDA0001796306540000041
wherein the content of the first and second substances,
d represents a nanobody resisting CD 47;
l3 represents no or a linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to CD 47.
In another preferred example, the Complementarity Determining Regions (CDRs) of the nanobody consist 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 nanobody are separated by 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 the bifunctional antibody of claim 1.
In another preferred example, the polynucleotide has a polynucleotide as shown in SEQ ID No. 17 or SEQ ID No. 19 encoding the L chain of the bifunctional antibody.
In another preferred embodiment, the polynucleotide has a polynucleotide encoding the H chain of the bifunctional antibody as shown in SEQ ID No. 20 or SEQ ID No. 21 or SEQ ID No. 22.
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 example, the purity of the purified and separated target antibody is greater than 90%, 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 tumor comprises a malignant tumor.
In another preferred embodiment, the tumor or solid tumor is selected from the group consisting of: lung cancer, gastric cancer, head and neck cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, large intestine cancer, adrenal gland tumor, 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/CD20 bispecific antibody of the present invention. As shown in the figure, six bispecific antibodies designed by the present invention consist of CD47 nanobody and rituximab.
Figure 2 is a flow cytometry assay for the binding ability of bispecific antibodies to cell surface CD 20. The results show that: the EC50 of the bispecific antibody A of the present application is 9.669ug/mL, the EC50 of the bispecific antibody B is 6.327ug/mL, the EC50 of the bispecific antibody C is 11.33ug/mL, and the EC50 of the control antibody rituximab is 5.669 ug/mL. Thus, bispecific antibody B better retained the ability to bind CD 20.
FIG. 3 is a result of flow cytometry to examine the cell-binding effect of bispecific antibodies on Raji (CD47 positive/CD 20 positive). The results indicate that the bispecific antibody B of the present application is capable of binding to both CD47 and CD20 on the cell surface simultaneously, and the binding capacity is similar to that of the control antibody, thus demonstrating that the bispecific antibody retains a good target binding activity.
Figure 4 is the IC50 results of flow cytometry detection of bispecific antibody B. The IC50 of the bispecific antibody B is 2.496ug/mL, and the IC50 of the CD47 nanobody Nb1902-Fc is 0.7673 ug/mL.
FIG. 5 shows the result of CCK8 testing for the proliferation inhibitory toxicity of bispecific antibodies against Raji cells. The detection result of the CCK8 kit shows that the bispecific antibody B and the control antibody rituximab have similar proliferation inhibition effect on Raji cells and good biological activity.
FIG. 6 is candidate dual-antibody B-mediated phagocytosis of Raji cells by macrophages in vitro. The results show that: the double antibody B can effectively promote phagocytosis of Raji cells by macrophages.
FIG. 7 is the agglutination of human red blood cells by candidate diabodies B. The results show that: the double antibody B does not cause agglutination of human erythrocytes.
FIG. 8 is the test of the efficacy of candidate dual-antibody B in Raji tumor model mice. The results show that: the tumor inhibition rate of the candidate double-antibody B reaches 92 percent, is far higher than that of rituximab (the tumor inhibition rate is 57 percent) and is better than that of a control nano antibody (the tumor inhibition rate is 81 percent), so that the double-antibody B disclosed by the invention can play a synergistic effect of double targets in the tumor inhibition process, and achieves a relatively ideal anti-tumor effect.
FIG. 9 is a graph showing the purity analysis of the anti-CD 47/CD20 bispecific antibody B of the present invention. SEC detection results of expressing purified bispecific antibody show that the purity of the bispecific antibody reaches 99.33%.
Figure 10 is a temperature stability test of candidate diabody B. The results show that: the double-antibody B shows good stability at room temperature of 25 ℃ and high temperature of 40 ℃, and the stability of the antibody is not influenced by the concentration. Therefore, the candidate double-antibody B is proved to be a double-antibody drug development candidate with good stability.
Detailed Description
The present inventors have made extensive and intensive studies and as a result, have unexpectedly found a bifunctional antibody comprising an anti-CD 20 antibody and an anti-CD 47 nanobody connected in series, which is a homodimer. In vitro experiments prove that the bifunctional antibody can be combined with CD20 and CD47 simultaneously, so that the bifunctional antibody can play a role in treating CD20 positive tumor cells (particularly malignant tumor cells), and 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", "nanobody" have the same meaning and refer to the cloning of the variable regions of the heavy chains of an antibody, creating a single domain antibody consisting of only one heavy chain variable region, which 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 form 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 (FRs). The variable regions of the native heavy and light chains each contain four FR regions, roughly in an β -fold configuration, connected by three CDRs forming a connecting loop, and in some cases may form part β -fold structures.
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, L2 and L3, wherein L1 links the heavy chain variable region VH of the anti-CD 47 nano antibody and the anti-CD 20 antibody of the invention, L2 links the light chain variable region VL of the anti-CD 47 nano antibody and the anti-CD 20 antibody of the invention, and L3 links the heavy chain constant region CH of the anti-CD 47 nano antibody and the anti-CD 20 antibody of the invention.
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, L2 and L3 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
The CD47 molecule, which is a highly glycosylated transmembrane protein of about 50kD, consists of an extracellular N-terminal immunoglobulin variable domain and 5 transmembrane segments with highly hydrophobic extensions and 1 short, selectively spliced cytoplasmic domain CD47 is widely distributed and expressed on the surface of myeloid cells such as macrophages, granulocytes, dendritic cells, mast cells and hematopoietic stem cells, and is highly expressed particularly in hematopoietic stem cells CD47 is a pluripotent molecule whose major function includes binding ① to CD47 ligand signaling regulatory protein (SIRP) α and interacting with Integrin, modulating intercellular communication between CD47 and SIRP α on the surface of macrophages, activating tyrosine phosphorylase, inhibiting the accumulation of myosin under the synaptic membrane, as an "separate" marker ", which interacts with the extracellular signaling protein, and is involved in the proliferation of various types of myeloid tumors, such as acute myeloid leukemia, chronic myeloproliferative leukemia, acute myeloproliferative leukemia, chronic myeloproliferative leukemia, acute myeloproliferative leukemia, chronic myeloproliferative leukemia, leukemia.
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 antibody of the invention, regardless of which end of the anti-CD 20 antibody is linked, is presented as a dimer by linking two identical anti-CD 47 antibodies by a linker. 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-CD 20 antibodies
The transmembrane protein on the B lymphocyte of the leukocyte differentiation antigen 20(CD20) exists in a non-glycosylated form, is a phosphorylated protein molecule with the molecular weight of 33-37KD, is positioned on the surface of the B lymphocyte, and is the differentiation antigen on the surface of the B lymphocyte. The conformation comprises four transmembrane regions (TM1-4), of which only the amino acid sequence between TM3 and TM4 is outside the cell membrane, TM1 and TM2 are linked, and the region between TM2 and TM3, the N-and C-termini, are inside the cytoplasm. CD20 has no known ligand, is part of a multi-somatic cell surface complex that regulates calcium transport, and is involved in regulating B cell activation and proliferation. Because of its expression in tumor cells of B cell-derived lymphomas, leukemias, and the like, and B cells involved in immune and inflammatory diseases, it has become a target for the treatment of lymphomas, leukemias, and certain autoimmune diseases. The anti-CD 20 antibody useful in the present invention consists of the light chain shown in SEQ ID No. 11 and the heavy chain shown in SEQ ID No. 12, with the corresponding light chain coding sequence as set forth in SEQ ID No.:17, heavy chain coding sequence is as shown in SEQ ID No.: 18, respectively.
Bifunctional antibodies (bispecific antibodies)
As used herein, the terms "bispecific antibody," "bifunctional antibody," "antibody of the invention," "dual anti," "dual-antibody," "bifunctional fusion antibody" are used interchangeably to refer to an anti-CD 20/CD47 bispecific antibody that binds CD20 and CD47 simultaneously.
In the present invention, the bifunctional antibody comprises:
(a) antibodies against CD 20; and
(b) a monovalent form of a nanobody against CD47 linked to the anti-CD 20 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 BDA0001796306540000121
wherein the content of the first and second substances,
each D is independently a nanobody that is absent or anti-CD 47, and at least one D is a nanobody that is anti-CD 47;
l1, L2, L3 are each independently a key or linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to CD 47.
In a preferred embodiment, the diabody of the present invention is formed by fusing an anti-CD 20 antibody and a nanobody 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 the peptide chains being linked by a disulfide bond, wherein any one pair of peptide chains has a structure of L chain and H chain shown in formula II from N-terminus to C-terminus:
Figure BDA0001796306540000131
wherein the content of the first and second substances,
d represents a nanobody resisting CD 47;
l3 represents no or a linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to CD 47.
In formula I or formula II, a preferred L chain is shown in SEQ ID NO. 11 or 13 and a preferred H chain is shown in SEQ ID NO. 14 or 15 or 16.
The sequence coding the L chain is shown in SEQ ID NO. 17 or 19, and the sequence coding the H chain is shown in SEQ ID NO. 20 or 21 or 22. (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 are antibodies having anti-CD 47 and anti-CD 20 activities, comprising two structures of formula I as described 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 BDA0001796306540000141
Figure BDA0001796306540000151
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 CD20, 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 nanobody (or its conjugate) of the present 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 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 be simultaneously combined with CD20 and CD47, the combination configuration of the bifunctional antibody and a combined target is kept unchanged, and the molecule is stable.
2. The bifunctional antibody can be expressed in HEK293F cells, the purity of the bifunctional antibody can reach 99.33% only by one-step affinity chromatography purification, and the preparation method is simple, convenient and feasible.
3. The bifunctional antibody can be effectively combined with double targets of CD47 and CD20, has good biological activity of the CD47 antibody, and perfectly maintains the biological activity of cetuximab.
4. The bifunctional antibody of the invention does not cause agglutination of human red blood cells and has better safety.
5. The bifunctional antibody of the invention has obvious anti-tumor effect in mice, has better anti-tumor effect than a single-target antibody, and shows the synergistic effect of double antibodies.
Therefore, the anti-CD 47/CD20 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/CD20 bispecific antibody molecule sequence design
The structure of the bispecific antibody provided by the invention, which can simultaneously bind to CD47 and CD20 extracellular domains, is shown in figure 1.CD47 nanobody sequences are derived from patent 201810151752.6; then the CD47 nano antibody is fused with the heavy chain of the rituximab, and the heavy chain is formed by Linker ((GGGGS)4) The rituximab heavy chain (LALA) was linked to the CD47 nanobody with the following amino acid sequence (the underlined sequence):
a: the amino acid sequence is shown as SEQ ID No. 14, the CD47 nano antibody is connected to the N end of the rituximab heavy chain through a linker (GGGGS) 4;
QVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
b: the amino acid sequence is shown as SEQ ID NO. 15, and the CD47 nano antibody is connected to the C end of the rituximab heavy chain through a linker (GGGGS) 4;
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGG GSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSS
c: the amino acid sequence is shown in SEQ ID NO. 16, and the CD47 nano antibody is simultaneously connected to the N end and the C end of the rituximab heavy chain through a linker (GGGGS) 4.
QVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSQVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSS
The light chain of the bispecific antibody molecule is:
a: a rituximab light chain represented by SEQ ID No. 11;
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
b: the CD47 nano antibody is connected to the N end of the rituximab light chain through a linker (GGGGS) 4.
QVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
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 ID No. 20, SEQ ID No. 21 and SEQ ID No. 22, and the base sequences of the light chain are shown as SEQ ID No. 17 and SEQ ID No. 19. The above base sequences were synthesized to expression vectors pCDNA3.1(+) respectively.
Example 2 anti-CD 47/CD20 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) respectively preparing a large amount of plasmids containing heavy chains and plasmids containing light chains by using an OMEGA plasmid answer kit, and filtering and sterilizing in a superclean bench 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% CO2Shaking tableCulturing in an incubator for 6 days; (4) centrifuging to obtain a supernatant, and combining the supernatant with protein A beads for 1h at room temperature; (5) washing away the foreign proteins and other impurities with phosphate buffer solution of pH7.0, eluting the target antibody protein with 0.1MpH3.0 of Glycine, and numbering the obtained anti-CD 47/CD20 antibody A-F; (6) ultrafiltering the eluted antibody into 1 × PBS solution, subpackaging and storing at-80 deg.C.
TABLE 1 CD47/CD20 bispecific antibody sequence composition
Figure BDA0001796306540000211
Example 3FACS detection of antibody binding to cell surface antigens
Binding of anti-CD 47/CD20 bispecific antibody (A-F) to cell surface CD 20: (1) will be 3X105Individual Raji (CD20+) cells (per sample) were incubated with 100uL of CD47 nanobody Nb1902(e.coli expression) at a concentration of 80ug/mL for 20min at 4 ℃; (2) antibody dilution: each group of antibodies was diluted in a gradient (100ug/mL, 50ug/mL, 25ug/mL, 12.5ug/mL, 6.25ug/mL, 3.125ug/mL, 1.56ug/mL, 0.78ug/mL, 0.39ug/mL, 0.195ug/mL, 0.0976ug/mL, 0.0488ug/mL), 100uL of the diluted antibodies were incubated with Raji cells per well for 20min, 4 ℃ and a positive control (rituximab) was set; (2) the cells were washed 2 times with PBS, added with anti-hFc-FITC from eBioscience (used at 1: 200 dilution), incubated at 4 ℃ for 20min, washed 2 times with PBS and detected with flow cytometry (BD FACS Calibur), and data processed using graphpad prism 6 software. As shown in fig. 2, the bispecific antibody A, B, C of the present application was able to bind to CD20 on the cell surface, wherein the binding ability of the dual antibody B was similar to that of rituximab, thus demonstrating that the bispecific antibody B retained a good CD20 target binding activity.
Example 4FACS detection of the ability of double anti-B to bind to both CD47 and CD20
The specific process is as follows: (1) taking 3X10 samples of each5Each Raji cell was placed in PBS buffer, and diluted anti-CD 47/CD20 bispecific antibody and control antibody were added (antibody dilution final concentration gradient of 20 ug/m)L), incubating for 20min at 4 ℃; (2) respectively adding the diluted CD47 nano antibody-biotin and rituximab-biotin into corresponding holes, discharging the mixture evenly, and incubating the mixture for 20min at 4 ℃; (3) 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 then detected using a flow cytometer (BD FACS Calibur) with data processing using graphpad prism 6 software. The results are shown in FIG. 3, where the diabody B binds both CD47 and CD 20.
Example 5FACS 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 then a gradient dilution of anti-CD 47/CD20 bispecific antibody and control antibody (final concentration gradient of antibody dilution 30ug/mL, 25ug/mL, 20ug/mL, 15ug/mL, 10g/mL, 5ug/mL, 3.3ug/mL, 2.5ug/mL, 1.7ug/mL, 1.3ug/mL, 0.85ug/mL, 0.65ug/mL) was added, 100uL of each sample was added, 2ug hPa (ECD) -Fc-Biotin (SIR) was added to all samples at the same time, and incubation was carried out at 4 ℃ for 20 min; (2) the cells were washed 2 times with PBS, SA-PE from eBioscience was added, incubated at 4 ℃ for 20min, and the cells were washed 2 times with PBS and then detected with a flow cytometer (BD FACS Calibur) and processed with the graphpad prism 6 software. The results are shown in FIG. 4, IC50 for bispecific antibody B was 2.496ug/mL, while IC50 for the control antibody (Nb1902-Fc) was 0.7673 ug/mL.
Example 6 CCK8 testing of the proliferation-inhibiting toxicity of diabody B on Raji cells
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) Trypsinize Raji cells, neutralize complete medium, wash once with PBS, 3 × 104Resuspended in a concentration of/ml (1640+ 1% FBS), and pipetted into 96-well plates at 100 ul/well, 37 ℃ and 5% CO2 for 24 hours. (3) The antibody (1640 in 1% FBS) was diluted 1h before 24h in cell culture with a gradient of 1E-6M, 2E-7M, 4E-8M, 8E-9M, 1.6E-9M. (4) The medium in the cells was aspirated and 100ul of diluted antibody was added. (5) After 72h, adding 10ul of CCK8 solution into each well, 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. 5 indicate that the inhibitory activity of the anti-CD 47/CD20 bispecific antibody of the present application on cell proliferation is the same as that of rituximab, which indicates that the bispecific antibody of the present application perfectly retains the biological activity of rituximab.
Example 7 candidate diabodies B mediate in vitro phagocytosis of Raji cells by macrophages
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) Centrifuging at 3000rpm and 4 deg.C for 4min, adding 200ul PBS/well, resuspending, and detecting by flow.
The results are shown in fig. 6, and indicate that the double antibody B can effectively promote phagocytosis of Raj i cells by macrophages.
Example 8 determination of agglutination of human Red blood cells by candidate diabodies B
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. CD47 nanobody Nb7 (a nanobody capable of causing human and monkey red blood cell agglutination in the laboratory), CD47 nanobody Nb1902 (derived from patent 201810151752.6), positive control antibody (B6H12) and negative control (IgG4) were diluted in a gradient (8000nM, 2000nM, 500nM, 125nM, 62.5nM, 15.625nM, 3.906nM and 0nM), respectively. 50uL of the diluted antibody was added to 50uL of human erythrocyte suspension (2%) and the reaction was carried out overnight at 37 ℃ before observing the results. The results show (FIG. 7) that candidate diabody B does not cause agglutination of erythrocytes. The results of the control antibody (B6H12) were consistent with those reported by Penkas. Petrova et al, 2016, Clin. cancer. Res.23 (4).
Example 9 detection of drug efficacy of candidate double-antibody B in Raji tumor model mice
Will contain 5x 106A 0.2mL cell suspension of Raji cells (cells suspended in DPBS) was inoculated subcutaneously in the right back of each mouse (NSG mice, 4-6 weeks). The average tumor volume reaches about 50-100mm3The grouped administration is started. Dose of antibody: 20 mg/kg. The administration was 1 time per day for 2 weeks.
The results are shown in fig. 8, and the results show that the tumor inhibition rate of the candidate double-antibody B reaches 92%, is far higher than that of rituximab (the tumor inhibition rate is 57%), and is better than that of a control nano antibody (the tumor inhibition rate is 81%), so that the double-antibody B of the invention plays a synergistic effect of double targets in the tumor inhibition process, and achieves a relatively ideal anti-tumor effect.
Example 10 purity analysis of candidate diabodies B
The purified sample, double antibody B, 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. 9, the purity of the double-antibody sample reaches 99.33%, and the double-antibody sample can be used for subsequent experiments.
Example 11 stability testing of candidate diabodies B
(1) Concentrating or diluting a sample with a total amount of 20mg to 10mg/mL, filtering the sample into a new centrifugal tube by using a 0.22um needle filter, filtering a sample preservative solution (1 × PBS, pH7.0) for dilution into a new centrifugal tube by using a 0.22um needle filter, subpackaging the filtered sample into 10 tubes into cryopreservation tubes by using 100uL of each tube, and marking according to the corresponding names of 10mg/mL in the following table; (2) adding 990uL of filtered sample preservation solution into a new centrifugal tube, then adding 110uL of filtered sample, fully and uniformly mixing, packaging 10 tubes into a freezing storage tube by 100uL of each tube, and marking according to the corresponding name of 1mg/mL in the following table; (3) 880uL of filtered sample preservation solution is added into a new centrifugal tube, then 220uL of filtered sample is added, the mixture is fully and uniformly mixed, each tube is 100uL, 10 tubes are subpackaged into a freezing storage tube, and the corresponding name mark is marked according to the following table 5 mg/mL; (4) placing the samples in corresponding incubators according to the following conditions:
influencing factor Conditions of standing Code
High temperature test 25℃±2℃ T1
Test for high Strength Damage 40℃±2℃ T2
A: high temperature test
Figure BDA0001796306540000251
B: test for high Strength Damage
Figure BDA0001796306540000252
(5) Sampling at corresponding detection time points, and performing on-machine detection.
The results are shown in FIG. 10 and show that: the double-antibody B shows good stability at room temperature of 25 ℃ and high temperature of 40 ℃, and the stability of the antibody is not influenced by the concentration. Therefore, the candidate double-antibody B is proved to be a double-antibody drug development candidate with good stability.
The above examples show that the anti-CD 47/CD20 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 bind to CD 47-positive and CD 20-positive Raj i cells and simultaneously block the cell surface CD47 interaction with SIRPa. The antibody can effectively play the phagocytosis of CD 47-mediated macrophages on tumor cells, and does not generate erythrocyte agglutination; in addition, the antibody can exert the synergistic effect of the double-target antibody in tumor inhibition in a mouse body, and has good stability. Therefore, the anti-CD 47/CD20 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/CD20 bispecific antibody and uses thereof
<130>P2018-0855
<160>22
<170>PatentIn version 3.5
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
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Lys
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405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
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Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
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Gly Tyr Ala Tyr Thr Ser Asp Cys Met Gly Trp Phe Arg Gln Thr Pro
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Gly Lys Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly Asn Ser
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Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp
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Asn Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
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Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys Ser Leu
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Arg Leu Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
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Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyTyr Ala Tyr Thr Ser Asp
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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 Gln Val
130 135 140
Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met
165 170 175
His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu TrpIle Gly Ala
180 185 190
Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly
195 200 205
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly
245 250 255
Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
260 265 270
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
275 280 285
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
290 295 300
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
305 310 315 320
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
325 330 335
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His LysPro
340 345 350
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
485 490 495
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
595 600 605
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
610 615 620
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
625 630 635 640
Ala Tyr Thr Ser Asp Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys
645 650 655
Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly Asn Ser Thr Asn
660 665 670
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser
675 680 685
Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
690 695 700
Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys Ser Leu Arg Leu
705 710 715 720
Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
725 730 735
<210>17
<211>639
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>17
cagatcgtgc tgtcccagtc ccccgccatc ctgtccgcct cccccggcga gaaggtgacc 60
atgacctgca gggcctcctc ctccgtgtcc tacatccact ggttccagca gaagcccggc 120
tcctccccca agccctggat ctacgccacc tccaacctgg cctccggcgt gcccgtgagg 180
ttctccggct ccggctccgg cacctcctac tccctgacca tctccagggt ggaggccgag 240
gacgccgcca cctactactg ccagcagtgg acctccaacc cccccacctt cggcggcggc 300
accaagctgg agatcaagag gaccgtggcc gccccctccg tgttcatctt ccccccctcc 360
gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaacaa cttctacccc 420
agggaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggag 480
tccgtgaccg agcaggactc caaggactcc acctactccc tgtcctccac cctgaccctg 540
tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600
tcctcccccg tgaccaagtc cttcaacagg ggcgagtgc 639
<210>18
<211>1353
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>18
caggtgcagc tgcagcagcc cggcgccgag ctggtgaagc ccggcgcctc cgtgaagatg 60
tcctgcaagg cctccggcta caccttcacc tcctacaaca tgcactgggt gaagcagacc 120
cccggcaggg gcctggagtg gatcggcgcc atctaccccg gcaacggcga cacctcctac 180
aaccagaagt tcaagggcaa ggccaccctg accgccgaca agtcctcctc caccgcctac 240
atgcagctgt cctccctgac ctccgaggac tccgccgtgt actactgcgc caggtccacc 300
tactacggcg gcgactggta cttcaacgtg tggggcgccg gcaccaccgt gaccgtgtcc 360
gccgcctcca ccaagggccc ctccgtgttc cccctggccc cctcctccaa gtccacctcc 420
ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480
tcctggaact ccggcgccct gacctccggc gtgcacacct tccccgccgt gctgcagtcc 540
tccggcctgt actccctgtc ctccgtggtg accgtgccct cctcctccct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggag 660
cccaagtcct gcgacaagac ccacacctgc cccccctgcc ccgcccccga ggccgccggc 720
ggcccctccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat ctccaggacc 780
cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 840
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccaggga ggagcagtac 900
aactccacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gtccaacaag gccctgcccg cccccatcga gaagaccatc 1020
tccaaggcca agggccagcc cagggagccc caggtgtaca ccctgccccc ctccagggac 1080
gagctgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta cccctccgac 1140
atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 1200
gtgctggact ccgacggctc cttcttcctg tactccaagc tgaccgtgga caagtccagg 1260
tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320
acccagaagt ccctgtccct gtcccccggc aag 1353
<210>19
<211>1065
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>19
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
ggcagccaga tcgtgctgtc ccagtccccc gccatcctgt ccgcctcccc cggcgagaag 480
gtgaccatga cctgcagggc ctcctcctcc gtgtcctaca tccactggtt ccagcagaag 540
cccggctcct cccccaagcc ctggatctac gccacctcca acctggcctc cggcgtgccc 600
gtgaggttct ccggctccgg ctccggcacc tcctactccc tgaccatctc cagggtggag 660
gccgaggacg ccgccaccta ctactgccag cagtggacct ccaacccccc caccttcggc 720
ggcggcacca agctggagat caagaggacc gtggccgccc cctccgtgtt catcttcccc 780
ccctccgacg agcagctgaa gtccggcacc gcctccgtgg tgtgcctgct gaacaacttc 840
taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaactcc 900
caggagtccg tgaccgagca ggactccaag gactccacct actccctgtc ctccaccctg 960
accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 1020
ggcctgtcct cccccgtgac caagtccttc aacaggggcg agtgc 1065
<210>20
<211>1779
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>20
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
ggcagccagg tgcagctgca gcagcccggc gccgagctgg tgaagcccgg cgcctccgtg 480
aagatgtcct gcaaggcctc cggctacacc ttcacctcct acaacatgca ctgggtgaag 540
cagacccccg gcaggggcct ggagtggatc ggcgccatct accccggcaa cggcgacacc 600
tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcctccacc 660
gcctacatgc agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgccagg 720
tccacctact acggcggcga ctggtacttc aacgtgtggg gcgccggcac caccgtgacc 780
gtgtccgccg cctccaccaa gggcccctcc gtgttccccc tggccccctc ctccaagtcc 840
acctccggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 900
accgtgtcct ggaactccgg cgccctgacc tccggcgtgc acaccttccc cgccgtgctg 960
cagtcctccg gcctgtactc cctgtcctcc gtggtgaccg tgccctcctc ctccctgggc 1020
acccagacct acatctgcaa cgtgaaccac aagccctcca acaccaaggt ggacaagaag 1080
gtggagccca agtcctgcga caagacccac acctgccccc cctgccccgc ccccgaggcc 1140
gccggcggcc cctccgtgtt cctgttcccc cccaagccca aggacaccct gatgatctcc 1200
aggacccccg aggtgacctg cgtggtggtg gacgtgtccc acgaggaccc cgaggtgaag 1260
ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 1320
cagtacaact ccacctacag ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg 1380
aacggcaagg agtacaagtg caaggtgtcc aacaaggccc tgcccgcccc catcgagaag 1440
accatctcca aggccaaggg ccagcccagg gagccccagg tgtacaccct gcccccctcc 1500
agggacgagc tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1560
tccgacatcg ccgtggagtg ggagtccaac ggccagcccg agaacaacta caagaccacc 1620
ccccccgtgc tggactccga cggctccttc ttcctgtact ccaagctgac cgtggacaag 1680
tccaggtggc agcagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1740
cactacaccc agaagtccct gtccctgtcc cccggcaag 1779
<210>21
<211>1779
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>21
caggtgcagc tgcagcagcc cggcgccgag ctggtgaagc ccggcgcctc cgtgaagatg 60
tcctgcaagg cctccggcta caccttcacc tcctacaaca tgcactgggt gaagcagacc 120
cccggcaggg gcctggagtg gatcggcgcc atctaccccg gcaacggcga cacctcctac 180
aaccagaagt tcaagggcaa ggccaccctg accgccgaca agtcctcctc caccgcctac 240
atgcagctgt cctccctgac ctccgaggac tccgccgtgt actactgcgc caggtccacc 300
tactacggcg gcgactggta cttcaacgtg tggggcgccg gcaccaccgt gaccgtgtcc 360
gccgcctcca ccaagggccc ctccgtgttc cccctggccc cctcctccaa gtccacctcc 420
ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480
tcctggaact ccggcgccct gacctccggc gtgcacacct tccccgccgt gctgcagtcc 540
tccggcctgt actccctgtc ctccgtggtg accgtgccct cctcctccct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggag 660
cccaagtcct gcgacaagac ccacacctgc cccccctgcc ccgcccccga ggccgccggc 720
ggcccctccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat ctccaggacc 780
cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 840
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccaggga ggagcagtac 900
aactccacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gtccaacaag gccctgcccg cccccatcga gaagaccatc 1020
tccaaggcca agggccagcc cagggagccc caggtgtaca ccctgccccc ctccagggac 1080
gagctgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta cccctccgac 1140
atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 1200
gtgctggact ccgacggctc cttcttcctg tactccaagc tgaccgtgga caagtccagg 1260
tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320
acccagaagt ccctgtccct gtcccccggc aagggcggcg gcggcagcgg cggcggcggc 1380
agcggcggcg gcggcagcgg cggcggcggc agccaggtgc agctgcagga gtccggcggc 1440
ggcctggtgc agcccggcgg ctccctgagg ctgtcctgcg ccgcctccgg ctacgcctac 1500
acctccgact gcatgggctg gttcaggcag acccccggca agggcctgga gggcgtggcc 1560
ctgatctaca cccccggcaa ctccaccaac tacgccgact ccgtgaaggg caggttcacc 1620
atctcccagg acaactccaa gtccaccgtg tacctgcaga tgaactccct gagggccgag 1680
gacaccgcca tgtactactg cgccgccagg aggggcgcct gctccctgag gctgcccttc 1740
ttctactggg gccagggcac cctggtgacc gtgtcctcc 1779
<210>22
<211>2205
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>22
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
ggcagccagg tgcagctgca gcagcccggc gccgagctgg tgaagcccgg cgcctccgtg 480
aagatgtcct gcaaggcctc cggctacacc ttcacctcct acaacatgca ctgggtgaag 540
cagacccccg gcaggggcct ggagtggatc ggcgccatct accccggcaa cggcgacacc 600
tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcctccacc 660
gcctacatgc agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgccagg 720
tccacctact acggcggcga ctggtacttc aacgtgtggg gcgccggcac caccgtgacc 780
gtgtccgccg cctccaccaa gggcccctcc gtgttccccc tggccccctc ctccaagtcc 840
acctccggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 900
accgtgtcct ggaactccgg cgccctgacc tccggcgtgc acaccttccc cgccgtgctg 960
cagtcctccg gcctgtactc cctgtcctcc gtggtgaccg tgccctcctc ctccctgggc 1020
acccagacct acatctgcaa cgtgaaccac aagccctcca acaccaaggt ggacaagaag 1080
gtggagccca agtcctgcga caagacccac acctgccccc cctgccccgc ccccgaggcc 1140
gccggcggcc cctccgtgtt cctgttcccc cccaagccca aggacaccct gatgatctcc 1200
aggacccccg aggtgacctg cgtggtggtg gacgtgtccc acgaggaccc cgaggtgaag 1260
ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 1320
cagtacaact ccacctacag ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg 1380
aacggcaagg agtacaagtg caaggtgtcc aacaaggccc tgcccgcccc catcgagaag 1440
accatctcca aggccaaggg ccagcccagg gagccccagg tgtacaccct gcccccctcc 1500
agggacgagc tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1560
tccgacatcg ccgtggagtg ggagtccaac ggccagcccg agaacaacta caagaccacc 1620
ccccccgtgc tggactccga cggctccttc ttcctgtact ccaagctgac cgtggacaag 1680
tccaggtggc agcagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1740
cactacaccc agaagtccct gtccctgtcc cccggcaagg gcggcggcgg cagcggcggc 1800
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcc aggtgcagct gcaggagtcc 1860
ggcggcggcc tggtgcagcc cggcggctcc ctgaggctgt cctgcgccgc ctccggctac 1920
gcctacacct ccgactgcat gggctggttc aggcagaccc ccggcaaggg cctggagggc 1980
gtggccctga tctacacccc cggcaactcc accaactacg ccgactccgt gaagggcagg 2040
ttcaccatct cccaggacaa ctccaagtcc accgtgtacc tgcagatgaa ctccctgagg 2100
gccgaggaca ccgccatgta ctactgcgcc gccaggaggg gcgcctgctc cctgaggctg 2160
cccttcttct actggggcca gggcaccctg gtgaccgtgt cctcc 2205

Claims (10)

1. A bifunctional antibody, wherein said bifunctional antibody comprises:
(a) antibodies against CD 20; and
(b) a monovalent form of a nanobody against CD47 linked to the anti-CD 20 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 FDA0001796306530000011
wherein the content of the first and second substances,
each D is independently a nanobody that is absent or anti-CD 47, and at least one D is a nanobody that is anti-CD 47;
l1, L2, L3 are each independently a key or linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to 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 FDA0001796306530000012
wherein the content of the first and second substances,
d represents a nanobody resisting CD 47;
l3 represents no or a linker element;
VL represents the light chain variable region of the anti-CD 20 antibody;
CL represents the light chain constant region of the anti-CD 20 antibody;
VH represents the heavy chain variable region of the anti-CD 20 antibody;
CH represents the heavy chain constant region of an anti-CD 20 antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to CD20 and binding to 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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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021218874A1 (en) * 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 Bispecific antibody targeting human claudin and human pdl1 proteins, and application thereof
CN113968909A (en) * 2020-07-22 2022-01-25 信达生物制药(苏州)有限公司 Method for purifying bispecific antibody
CN115607690A (en) * 2021-07-15 2023-01-17 广东精观生物医药科技有限公司 Molecular probe and preparation method and application thereof
WO2023056969A1 (en) * 2021-10-09 2023-04-13 Hutchmed Limited Bispecific antibodies specifically binding to cd47 and cd20, and uses thereof

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Publication number Priority date Publication date Assignee Title
CN105121467A (en) * 2012-12-03 2015-12-02 诺夫免疫股份有限公司 Anti-CD47 antibodies and methods of use thereof
WO2016020065A1 (en) * 2014-08-08 2016-02-11 Ludwig-Maximilians-Universität Subcutaneously administered bispecific antibodies for use in the treatment of cancer
CN105622753A (en) * 2014-11-04 2016-06-01 博生吉医药科技(苏州)有限公司 PD-1 monoclonal antibody and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105121467A (en) * 2012-12-03 2015-12-02 诺夫免疫股份有限公司 Anti-CD47 antibodies and methods of use thereof
WO2016020065A1 (en) * 2014-08-08 2016-02-11 Ludwig-Maximilians-Universität Subcutaneously administered bispecific antibodies for use in the treatment of cancer
CN105622753A (en) * 2014-11-04 2016-06-01 博生吉医药科技(苏州)有限公司 PD-1 monoclonal antibody and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021218874A1 (en) * 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 Bispecific antibody targeting human claudin and human pdl1 proteins, and application thereof
CN113968909A (en) * 2020-07-22 2022-01-25 信达生物制药(苏州)有限公司 Method for purifying bispecific antibody
CN115607690A (en) * 2021-07-15 2023-01-17 广东精观生物医药科技有限公司 Molecular probe and preparation method and application thereof
CN115607690B (en) * 2021-07-15 2023-12-15 广东精观生物医药科技有限公司 Molecular probe and preparation method and application thereof
WO2023056969A1 (en) * 2021-10-09 2023-04-13 Hutchmed Limited Bispecific antibodies specifically binding to cd47 and cd20, and uses thereof

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Denomination of invention: Anti CD47/CD20 bispecific antibody and its use

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