anti-CD 47/CD20 bispecific antibodies and uses thereof
阅读说明：本技术 抗cd47/cd20双特异性抗体及其用途 (anti-CD 47/CD20 bispecific antibodies and uses thereof ) 是由 万亚坤 朱敏 沈晓宁 于 2018-09-11 设计创作，主要内容包括：本发明涉及抗CD47/CD20双特异性抗体及其用途。具体地,本发明提供了抗CD47/CD20双特异性抗体,包括：(a)抗CD20的抗体(如利妥昔单抗)；和(b)与所述抗CD20的抗体相连接的单价形式的抗CD47的纳米抗体。本发明的双功能抗体具有良好的稳定性,不仅能够特异地靶向CD47和CD20,有效阻断CD47与其配体SIRPa的相互作用,同时有效结合细胞表面CD20,还能显著抑制肿瘤细胞增殖,实现了双功能抗体的协同作用,具有良好的应用前景。(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.)
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:
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:
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.
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
(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:
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
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
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:
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
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
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.
Drawings
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
FIG. 3 is a result of flow cytometry to examine the cell-binding effect of bispecific antibodies on Raji (CD47 positive/
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
(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:
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:
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
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.
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