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CN110835375A - anti-PD-1/EGFR bispecific antibody and application thereof - Google Patents

anti-PD-1/EGFR bispecific antibody and application thereof Download PDF

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CN110835375A
CN110835375A CN201811088993.7A CN201811088993A CN110835375A CN 110835375 A CN110835375 A CN 110835375A CN 201811088993 A CN201811088993 A CN 201811088993A CN 110835375 A CN110835375 A CN 110835375A
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万亚坤
朱敏
沈晓宁
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Shanghai Luoqi Bio Pharmaceutical Technology Co Ltd
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Abstract

The invention provides an anti-PD-1/EGFR bispecific antibody and application thereof, in particular to a bifunctional antibody, which comprises: (a) antibodies against Epidermal Growth Factor (EGFR); and (b) a bivalent form of a nanobody against PD-1 linked to the anti-Epidermal Growth Factor (EGFR) antibody. The bifunctional antibody of the invention can be combined with EGFR and PD-1 simultaneously, thereby playing a role in treating EGFR positive tumor cells (especially malignant tumor cells).

Description

anti-PD-1/EGFR bispecific antibody and application thereof
Technical Field
The invention belongs to the field of tumor immunology, and particularly relates to an anti-PD-1/EGFR bispecific antibody and application thereof.
Background
Programmed cell death protein 1(PD-1) is a member of the CD28 superfamily. As a T cell inhibitory receptor, the T cell inhibitory receptor can limit the functions of T cell effectors in tumor cells and has an important role in tumor immune escape. PD-1 is highly expressed in tumor infiltrating lymphocytes, tumor specific T cells and some injury related T cells, and the ligand PD-L1 is highly expressed on the surfaces of various tumors, when the PD-L1 of the tumor cells is combined with the PD-1 on the surface of the T cells, inhibitory signals are formed, T cell apoptosis is induced, and activation and proliferation of the T cells are inhibited, so that the tumor cells are prevented from being attacked by the T cells, and tumor immune escape is formed. Therefore, blocking the interaction of PD-1 and PD-L1 can effectively restore the killing function of T cells to tumors. The broad-spectrum anti-tumor function and good clinical performance of the PD-1 antibody are well-evaluated in the industry, and breakthrough progress is made in the treatment process of non-small cell lung cancer, ovarian cancer, renal cell carcinoma, melanoma, leukemia and the like. Particularly, after the unprecedented clinical efficacy data is revealed in 2013 by American Society for Clinical Oncology (ASCO), the medicine becomes the most popular and new medicine in research in the global pharmaceutical industry.
In addition, the tumor cell surface antigen Epidermal Growth Factor Receptor (EGFR) is a glycoprotein, widely distributed on the surfaces of Epithelial cells, glial cells, keratinocytes, fibroblasts and the like except vascular tissues in mammals, and is involved in important physiological processes such as cell growth, proliferation, differentiation and the like. EGFR is highly expressed or abnormally expressed in most solid tumors, and the overexpression of the EGFR is related to the inhibition of the proliferation, angiogenesis, tumor metastasis and apoptosis of tumor cells and is a hot target point for tumor treatment.
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, the anti-tumor double antibody which has stable structure, good specificity and easy preparation is urgently developed in the field.
The bispecific antibody development of the invention is a double-antibody technology based on the nano antibody, the nano antibody has small and stable molecules, and the stability change of the antibody can not be caused after the fusion with the monoclonal antibody, thereby having great advantages for the subsequent process development.
Disclosure of Invention
The invention aims to provide an anti-tumor double antibody which has stable structure and good specificity and is easy to prepare.
In a first aspect, the present invention provides a bifunctional antibody comprising:
(a) antibodies against Epidermal Growth Factor (EGFR); and
(b) a bivalent form of a nanobody against PD-1 linked to the anti-Epidermal Growth Factor (EGFR) antibody.
In another preferred embodiment, the anti-Epidermal Growth Factor (EGFR) antibody and the anti-PD-1 nanobody are linked by a linker peptide; preferably, the linker peptide comprises an antibody constant region sequence.
In another preferred embodiment, the nanobody against PD-1 is linked to a region of the antibody against Epidermal Growth Factor (EGFR) 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 against PD-1 is linked to the beginning of the heavy chain variable region, and/or the light chain variable region of the antibody against Epidermal Growth Factor (EGFR).
In another preferred example, the nanobody against PD-1 is linked to the beginning of the light chain variable region of the antibody against Epidermal Growth Factor (EGFR).
In another preferred embodiment, the nanobody against PD-1 is linked to the end of the heavy chain constant region of the antibody against Epidermal Growth Factor (EGFR).
In another preferred embodiment, the number of the nanobodies against PD-1 is 1 to 6, preferably 2 to 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 BDA0001803880620000031
wherein,
each D is independently a nanobody against PD-1 or no, and at least one D is a nanobody against PD-1;
l1, L2, L3, L4, L5, L6 are each independently a bond or a linker element;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
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 example, D in "D-L1-D-L2" are all nanobodies against PD-1 (corresponding to antibodies D, E and F).
In another preferred embodiment, the tab members may be identical or different.
In another preferred example, the L1, L2, L3, L4, L5 or L6 are each independently selected from GS, GGGGS (SEQ ID No.:8), GGGGSGGGS (SEQ ID No.:9), ggggsggggsggsggggs (SEQ ID No.: 10).
In another preferred example, the VL has a CDR1 as shown in SEQ ID No. 23, a CDR2 as shown in SEQ ID No. 24 and a CDR3 as shown in SEQ ID No. 25.
In another preferred example, the VH has CDR1 as shown in SEQ ID No. 26, CDR2 as shown in SEQ ID No. 27 and CDR3 as shown in SEQ ID No. 28.
In another preferred example, the VL has an amino acid sequence as set forth in SEQ ID No. 29.
In another preferred embodiment, the VH has an amino acid sequence as shown in SEQ ID No. 30.
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. 16.
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. 13 or SEQ ID No. 14 or SEQ ID No. 15.
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. 22.
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. 19 or SEQ ID No. 20 or SEQ ID No. 21.
In another preferred embodiment, the bifunctional antibody has a binding affinity (KD) for EGFR of at least 2E-09M, preferably 3E-09M.
In another preferred embodiment, the bifunctional antibody has a binding affinity (KD) for PD-1 of at least 1.5E-08M, preferably 5E-08M.
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 further comprises an active fragment and/or derivative of the bifunctional antibody, wherein the active fragment and/or derivative retains 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the anti-EGFR activity and 70-100% of the anti-PD-1 activity of the bifunctional antibody.
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 "D-L6-D-L5" is nothing.
In another preferred embodiment, the bifunctional antibody has a structure represented by formula II from N-terminus to C-terminus:
Figure BDA0001803880620000051
wherein,
each D represents a nano antibody against PD-1;
l1, L2, L3, L4 represent no or linker elements, respectively;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
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 a bifunctional antibody according to the first aspect of the invention.
In another preferred example, the polynucleotide has a polynucleotide as shown in SEQ ID No. 17 or SEQ ID No. 22 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. 19 or SEQ ID No. 20 or SEQ ID No. 21.
In another preferred embodiment, the ratio of the polynucleotide encoding the L chain to the polynucleotide encoding the H chain is 3:2 or 1:1, preferably 3:2, in said polynucleotides.
In a third aspect, the present invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the vector contains all of the polynucleotides of the second aspect of the invention simultaneously.
In another preferred embodiment, the vectors each comprise a polynucleotide of the polynucleotides of the second aspect of the invention.
In another preferred embodiment, the vector is an expression vector.
In another preferred embodiment, the vector comprises a plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.
In a fourth aspect, the present invention provides a genetically engineered host cell comprising a vector or genome according to the third aspect of the invention into which a polynucleotide according to the second aspect of the invention has been integrated.
In a fifth aspect, the present invention provides a method of producing an antibody according to the first aspect of the invention, comprising the steps of:
(i) culturing the host cell of the fourth aspect of the invention under suitable conditions to obtain a mixture comprising the antibody of the first aspect of the invention;
(ii) (ii) purifying and/or separating the mixture obtained in step (i) to obtain the antibody according to the first aspect of the invention.
In another preferred example, the purification can be performed by protein a affinity column purification and separation to obtain the target antibody.
In another preferred embodiment, the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
In a sixth aspect, the present invention provides a pharmaceutical composition comprising:
(I) a bifunctional antibody according to the first aspect of the invention; and
(II) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises an additional 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 is a solid tumor.
In another preferred embodiment, the tumor is a malignant tumor.
In another preferred embodiment, the tumor is selected from the group consisting of: malignant melanoma, non-small cell lung cancer, renal cancer, head and neck squamous carcinoma, bladder cancer, or a combination thereof.
The present invention also provides a method of treating a tumor comprising the steps of: administering to a subject in need thereof a safe and effective amount of an antibody according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention.
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-PD-1/EGFR bispecific antibody of the present invention. As shown in the figure, the six bispecific antibodies designed by the invention consist of a PD-1 nano antibody bivalent body and cetuximab.
FIG. 2 is a SDS-PAGE pattern of the anti-PD-1/EGFR bispecific antibody of the present invention.
FIG. 3 is the result of flow cytometry to detect the blocking effect of bispecific antibody on PD-1/PD-L1. The result shows that the candidate double antibody D, E, F has better blocking activity.
Figure 4 is the results of flow cytometry to detect the binding effect of bispecific antibodies on BxPC3(EGFR positive) cells. The results show that the bispecific antibody F of the present application is capable of binding to EGFR on the cell surface and the binding capacity is similar to that of cetuximab, thus demonstrating that the bispecific antibody retains a good target binding activity.
Figure 5 is the IC50 results of flow cytometric detection of bispecific antibodies. The bispecific antibody of the present application had an IC50 of 2.128ug/mL and the control antibody Opdivo had an IC50 of 2.898 ug/mL. Thus, it was shown that bispecific antibody F retained better PD-1/PD-L1 blocking activity.
FIG. 6 shows the results of the affinity assay of bispecific antibodies to human PD-1 molecules. Bispecific antibody F had an affinity for PD-1 of 1.55E-8M.
Figure 7 is the results of the affinity assay of bispecific antibodies to human EGFR molecules. Bispecific antibody F had an affinity for EGFR of 2.10E-9M.
FIG. 8 shows the results of activity detection of PD-1 antibody in bispecific antibody. The detection result of the PD-1/PD-L1 reporter gene detection system shows that the candidate bispecific antibody F has similar biological activity with the control antibody Opdivo.
Fig. 9 is the result of CCK8 testing for proliferation inhibitory toxicity of bispecific antibodies against a431 cells. The detection result of the CCK8 kit shows that the candidate bispecific antibody F and the control antibody cetuximab have similar proliferation inhibition effects on EGFR positive cells A431, and the biological activity of the EGFR antibody is perfectly maintained.
FIG. 10 is a SEC analysis chart of a particularly preferred anti-PD-1/EGFR bispecific antibody F of the present invention. SEC detection results of expressing the purified bispecific antibody show that the purity of the double anti-F (with the structure of the formula II) can reach 98.05 percent after one-step purification.
Detailed Description
The present inventors have made extensive and intensive studies and as a result, have unexpectedly obtained a bifunctional antibody comprising an anti-EGFR antibody and an anti-PD-1 nanobody in tandem. Preferably, the bifunctional antibodies of the present invention are homodimers. In vitro experiments prove that the bifunctional antibody can be combined with EGFR and PD-1 simultaneously so as to play a role in treating EGFR positive tumor cells (particularly malignant tumor cells), and therefore, the bifunctional antibody can be developed into an antitumor drug with excellent curative effect. On this basis, the present inventors have completed the present invention.
Term(s) for
Generally, an "antibody," also referred to as an "immunoglobulin," can be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ (l) and κ (k). There are five major heavy chain species (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain comprises a different sequence domain. The light chain comprises two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity for an antigen. The constant domains of the light Chain (CL) and heavy Chain (CH) confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light and one heavy chain. The specificity of an antibody depends on the structural complementarity of the antibody binding site and the epitope. The antibody binding site consists of residues derived primarily from the hypervariable region or Complementarity Determining Region (CDR). Occasionally, residues from non-highly variable or Framework Regions (FR) affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, otherwise designated as CDRs 1-L, CDR2-L, CDR3-L and CDRs 1-H, CDR2-H, CDR 3-H. Conventional antibody antigen binding sites therefore include six CDRs, comprising a collection of CDRs from each heavy and light chain v region.
As used herein, the terms "single domain antibody", "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 "affinity" is theoretically defined by an equilibrium association between an intact antibody and an antigen. The affinity of the diabodies of the invention may be assessed or determined by KD values (dissociation constants) (or other means of determination), for example by biofilm layer interference techniques (Bio-layer interference BLI) using FortebioRed96 instrument measurements.
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, L3 and L4, wherein the linker L1 and L4 connect two anti-PD-1 antibodies, L2 connects the dimer of the double anti-heavy chain anti-PD-1 antibody of the invention with the light chain variable region VL of the anti-EGFR antibody, and L3 connects the dimer of the double anti-light chain anti-PD-1 antibody of the invention with the heavy chain constant region CH of the anti-EGFR antibody.
Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element that is desired to be implemented in the linker. . One preferred joint is as follows:
L1L2, L3, L4, L5 and L6 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-PD-1 antibodies
Programmed cell death protein 1(PD-1) is a member of the CD28 superfamily. As a T cell inhibitory receptor, the T cell inhibitory receptor can limit the functions of T cell effectors in tumor cells and has an important role in tumor immune escape. PD-1 is highly expressed in tumor infiltrating lymphocytes, tumor specific T cells and some injury related T cells, and the ligand PD-L1 is highly expressed on the surfaces of various tumors, when the PD-L1 of the tumor cells is combined with the PD-1 on the surface of the T cells, inhibitory signals are formed, T cell apoptosis is induced, and activation and proliferation of the T cells are inhibited, so that the tumor cells are prevented from being attacked by the T cells, and tumor immune escape is formed. Therefore, blocking the interaction of PD-1 and PD-L1 can effectively restore the killing function of T cells to tumors. The broad-spectrum anti-tumor function and good clinical performance of the PD-1 antibody are well-evaluated in the industry, and breakthrough progress is made in the treatment process of non-small cell lung cancer, ovarian cancer, renal cell carcinoma, melanoma, leukemia and the like.
The sequence of the anti-PD-1 antibody of the present invention can be obtained by using nanobodies known or prepared by conventional methods or developed by screening or display techniques, such as the antibody described in patent application CN201610826020.3, and the skilled person can also modify or modify the anti-PD-1 antibody of the present 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-PD-1, and obtaining the modified or modified result by conventional determination methods.
Preferably, the anti-PD-1 antibody of the invention, regardless of which end of the anti-EGFR antibody is linked, is linked to two identical anti-PD-1 antibodies by a linker so as to appear as a dimer.
In another preferred embodiment, the bispecific antibody of the invention has a structure represented by formula II.
The anti-PD-1 antibody dimer can be obtained by expression of HEK293 cells or CHO cells.
The anti-PD-1 antibodies of the invention bind to mammalian PD-1, preferably human PD-1.
In the double antibody of the present invention, the binding affinity of the anti-PD-1 antibody of the present invention to PD-1 is 1.55E-08M, preferably not less than 5E-08M.
In another preferred embodiment, the bispecific antibody of the invention has a molecular weight of 200-500kD, for example a molecular weight of about 262 kD.
anti-EGFR antibodies
The tumor cell surface antigen Epidermal Growth Factor Receptor (EGFR) is a glycoprotein, widely distributed on the surfaces of Epithelial cells, glial cells, keratinocytes, fibroblasts and the like of blood vessel tissues in mammals, and is involved in important physiological processes such as cell growth, proliferation, differentiation and the like. EGFR is highly expressed or abnormally expressed in most solid tumors, and the overexpression of the EGFR is related to the inhibition of the proliferation, angiogenesis, tumor metastasis and apoptosis of tumor cells and is a hot target point for tumor treatment. The anti-EGFR antibody that can be used in the present invention consists of the amino acid sequence of the light chain shown in SEQ ID No. 11 and the amino acid sequence of the heavy chain shown in SEQ ID No. 12. The anti-EGFR antibody useful in the present invention consists of the nucleotide sequence of the light chain shown in SEQ ID No. 17 and the nucleotide sequence of the heavy chain shown in SEQ ID No. 18.
Bifunctional antibodies (bispecific antibodies)
As used herein, the terms "bispecific antibody", "diabody", "antibody of the invention", "diabody" and "bifunctional fusion antibody" are used interchangeably and refer to an anti-PD-1/EGFR bispecific antibody that binds both PD-1 and EGFR.
In the present invention, the bifunctional antibody comprises:
(a) antibodies against Epidermal Growth Factor (EGFR); and
(b) a bivalent form of a nanobody against PD-1 linked to the anti-Epidermal Growth Factor (EGFR) 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 BDA0001803880620000121
each D is independently a nanobody against PD-1 or no, and at least one D is a nanobody against PD-1;
l1, L2, L3, L4, L5, L6 are each independently a bond or a linker element;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
In a preferred embodiment, the diabody of the present invention is fused by an Epidermal Growth Factor (EGFR) antibody and a nanobody against programmed death factor 1(PD-1), and has two pairs of peptide chains symmetrical to each other, each pair of peptide chains comprising a light chain L chain and a heavy chain H chain, all of which are linked by a disulfide bond, wherein any one pair of peptide chains has a structure of L chain and H chain shown in formula II from N-terminus to C-terminus:
Figure BDA0001803880620000131
wherein,
each D represents a nano antibody against PD-1;
l1, L2, L3, L4 represent no or linker elements, respectively;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
In formula I or formula II, a preferred L chain is shown in SEQ ID No. 16, and a preferred H chain is shown in SEQ ID No. 14.
The sequence of the coding L chain is shown in SEQ ID NO. 22, and the sequence of the coding H chain is shown in SEQ ID NO. 20. (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 antibody of the invention refers to an antibody which has anti-PD-1 and anti-EGFR activities and comprises two structures shown in the formula I. 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 BDA0001803880620000141
Figure BDA0001803880620000151
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 bispecific antibody or active fragment thereof or fusion protein thereof of the present invention as described above, 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 PD-L1 protein molecules or EGFR, 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 EGFR and PD-1, and keeps the combination configuration of the bifunctional antibody and a combined target unchanged, and the molecule is stable.
2. The bifunctional antibody can be efficiently expressed in HEK293F cells, and can be efficiently purified only by one-step affinity chromatography, and the purity of the obtained antibody can reach 98% or higher.
3. The bifunctional antibody has high binding affinity with a binding target and good blocking activity. Even higher than EGFR and PD-1 binding affinity and blocking activity alone.
4. The bifunctional antibody not only has good biological activity of the PD-1 antibody, but also perfectly maintains the biological activity of the cetuximab.
5. The preparation method is simple and feasible.
The anti-PD-1/EGFR 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-PD-1/EGFR bispecific antibody molecule sequence design
The structure of the bispecific antibody which can simultaneously bind PD-1 and EGFR extracellular domain provided by the invention is shown in figure 1. The PD-1 nanobody sequence was as described in patent application CN201610826020.3, and PD-1 nanobody VHH sequences were connected in series via a Linker (GGGGSGGGS) to form a PD-1 nanobody dimer configuration, with specific amino acid sequences as follows (the underlined part is the Linker sequence):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS(SEQ ID No.:31)
then the PD-1 nano antibody dimer is respectively fused with a heavy chain and a light chain of the cetuximab, the heavy chain connects a cetuximab heavy chain (LALA) and the PD-1 nano antibody dimer by a linker ((GGGGS)4), and the connection method is as follows:
a: the amino acid sequence is shown as SEQ ID NO. 13, and the PD-1 nano antibody bivalent body is connected to the N end of a cetuximab heavy chain through a linker (GGGGS) 4;
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.:13)
b: the amino acid sequence is shown as SEQ ID No. 14, and the PD-1 nano antibody bivalent body is connected to the C end of a cetuximab heavy chain through a linker (GGGGS) 4;
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGG GGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS(SEQ ID No.:14)
c: the amino acid sequence is shown as SEQ ID NO. 15, and the PD-1 nano antibody bivalent body is simultaneously connected with the N end and the C end of a cetuximab heavy chain through a linker (GGGGS) 4.
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSG GGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS(SEQ ID No.:15)
The light chain of the bispecific antibody molecule is:
a: a cetuximab light chain represented by seq id No. 11;
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNo.:11)
b: the antibody consists of a PD-1 nano antibody bivalent body sequence and a cetuximab light chain sequence, wherein the amino acid sequence of the antibody is shown as SEQ ID NO. 16, and the PD-1 nano antibody bivalent body is connected to the N end of the cetuximab light chain through a linker (GGGGS) 4.
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID No.:16)
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. 19, SEQ ID No. 20 and SEQ ID No. 21, and the base sequences of the light chain are shown as SEQ ID No. 17 and SEQ ID No. 22. The above base sequences were synthesized to expression vectors pCDNA3.1(+) respectively.
Example 2: anti-PD-1/EGFR bispecific antibody expression purification
The plasmid where the synthetic gene is located is 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% CO2Culturing for 6 days in a shaking incubator; (4) centrifuging to obtain a supernatant, and combining the supernatant with protein A beads for 1h at room temperature; (5) washing away the foreign proteins and other impurities by using a phosphate buffer solution with pH7.0, then eluting the target antibody protein by using 0.1M Glycine with pH3.0, and numbering the obtained anti-PD-1/EGFR antibodies as shown in Table 1, wherein the anti-PD-1/EGFR antibodies respectively correspond to the A-F antibody structures shown in figure 1; (6) the eluted antibody was ultrafiltered into 1 XPBS solution and sampled for SDS-PAGE analysis.
As a result, as shown in FIG. 2, antibodies A to F shown in Table 1 were successfully prepared.
The prepared antibodies are respectively subpackaged and stored at-80 ℃.
TABLE 1 PD-1/EGFR bispecific antibody sequence composition (see FIG. 1 for Structure)
Figure BDA0001803880620000211
Figure BDA0001803880620000221
Example 3: FACS detection of blocking Activity of candidate double antibodies against PD-1/PD-L1
Blocking activity of candidate anti-PD-1/EGFR bispecific antibodies against PD-1/PD-L1: (1) taking 3X10 samples of each5293T/PD-1 stable cells in PBS buffer, adding diluted anti-PD-1/EGFR bispecific antibody, negative control antibody (AF70-Fc, a nanobody Fc fusion protein) and positive antibody (PD-1 nanobody dimer Fc fusion protein), antibody concentration of 1.67ug/mL and 6.67ug/mL, each sample 100uL, all samples simultaneously adding 2ug hPD-L1(ECD) -Fc-Biotin, 4 ℃ incubation 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 data using the software graphpad prism 6.
The results are shown in fig. 3, where the blocking activity of candidate diabody A, B, C is significantly reduced compared to the positive control antibody; and the candidate diabody D, E, F well retains the blocking activity (MFI is 37-65) on PD-1/PD-L1, so that the diabody D, E, F is selected for subsequent research.
Example 4: FACS detection of binding of candidate antibodies to cell surface antigens
Binding of candidate anti-PD-1/EGFR bispecific antibody to cell surface EGFR: (1) 100uL of the antibody with the concentration of 100ug/mL and 20ug/mL is respectively mixed with 3x105BxPC3 cells (with high EGFR expression) are incubated for 20min at 4 ℃, and a positive control (cetuximab) and a negative control (AF70-Fc, a nano antibody Fc fusion protein) are arranged at the same time; (2) PBS washing 2 times cell, adding eBioscienanti-hFc-FITC at ce (used at 1: 200 dilution), incubation at 4 ℃ for 20min, washing of the cells 2 times with PBS followed by detection with flow cytometry (BD FACS Calibur), and data processing using graphpad prism6 software.
As shown in fig. 4, the bispecific antibody F of the present invention can bind to EGFR on the cell surface, and the binding ability is similar to that of cetuximab, and better retains the target binding activity; whereas candidate diabodies D and E have reduced binding activity to cell surface EGFR. Bispecific antibody F was therefore selected for subsequent studies.
Example 5: FACS detection of antibody IC50
The specific process is as follows: (1) taking 3X10 samples of each5293T/PD-1 stable cells in PBS buffer, gradient dilution of anti-PD-1/EGFR bispecific antibody F and control antibody (antibody dilution final concentration gradient of 20ug/mL, 13.3ug/mL, 6.67ug/mL, 3.33ug/mL, 2.22ug/mL, 1.67ug/mL, 1.11ug/mL, 0.83ug/mL, 0.67ug/mL, 0.56ug/mL, 0.42ug/mL, 0.28ug/mL) were added, each sample was 100uL, all samples were added with 2ug hPD-L1(ECD) -Fc-Biotin at the same time, 4 ℃ incubation 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 data using the software graphpad prism 6.
The results are shown in FIG. 5, IC50 for bispecific antibody F was 2.128ug/mL, while IC50 for the control antibody (Opdivo) was 2.898 ug/mL.
Example 6: antibody affinity assay
6.1 binding to human PD1
The binding kinetics of the diabody F obtained in example 3 against recombinant human PD1 was measured by biofilm layer interference technique (Bio-layer interference BLI) using Fortebio Red96 instrument. For kinetic measurements, the double antibody F was diluted to 4ug/mL with PBST buffer; PD1 antigen (expressed by HEK293F, as in example two, with removal of Fc-tagged protein after cleavage by TEV enzyme) was diluted with PBST buffer in 1.5-fold gradients (200nM, 133.3nM, 88.9nM, 59.3nM, 39.5nM, 26.3nM), setting the instrument operating conditions: the temperature was 30 ℃ and Shake speed 1000 rpm. The antibody was captured with a Protein A-coated probe (Fortebio Part No:18-5010) for 180 s; binding the antigen with gradient dilution for 180 s; dissociation time 300 s; 10mM glycine (pH1.7) was regenerated 3 times for 5s each time. Analysis was performed using Fortebio Analysis version 9.0, 1:1 binding model Global model fitting was performed and the association rate (Kon), dissociation rate (Kdis) and dissociation constant KD were calculated.
The results are shown in FIG. 6, where the affinity of the diabody F for human PD-1 is 1.55E-8M.
6.2 binding to human EGFR
Similarly, the binding kinetics of the obtained double anti-F against recombinant human EGFR were measured by biofilm layer interference technique (Bio-layer interference BLI) using FortebioRed96 instrument. For kinetic measurements, the double antibodies F were diluted to 5ug/mL in PBST buffer, EGFR antigen (expressed by HEK293F, as in example two, with removal of Fc-tagged protein after cleavage by TEV enzyme) was diluted in PBST buffer in six concentration gradients (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) in 2-fold gradients, and the instrument operating conditions were set: the temperature was 30 ℃ and Shake speed 1000 rpm. The antibody was captured using a Protein A-coated probe (Fortebio Part No:18-5010) for a capture time of 60 s; binding the antigen with gradient dilution for 180 s; dissociation time 240 s; 10mM glycine (pH1.7) was regenerated 2 times for 5s each time. Analysis was performed using fortebioanalystiss version 9.0, 1:1 binding model Global model fitting was performed and the association rate (Kon), dissociation rate (Kdis) and dissociation constant KD were calculated.
The results are shown in FIG. 7, where the dual anti-F has an affinity for human EGFR of 2.10E-9M.
Example 7: luciferase reporter gene system for detecting activity of PD-1 antibody in bispecific antibody
The conventional competitive ELISA method can only reflect the antigen binding capacity and cannot reflect the mechanism of action of the drug. Mixed Lymphocyte Reaction (MLR) can detect biological activity by detecting secretion of IFN-gamma, but the detection period is too long and takes 4-5 days. And these methods require the use of primary cells, due to the complexity of the procedures and their origin from different individualsThe variability of the measurements was large. Detection methods based on T cell proliferation, cytotoxicity, etc. are not suitable as general biological activity detection methods, also because of the need to use primary cells and complicated procedures. The PD-1/PD-L1 reporter gene detection system (purchased from Nanjing Kingsler Biotechnology Co., Ltd.) can be used in the links of biological sample batch release, stability detection, process development and the like, and is used for judging the biological activity of PD-1 antibody drugs. When the added PD-1 antibody effectively blocks the interaction between PD-1 on the surface of an effector cell (GS-J2/PD-1) and PD-L1 on the surface of a target cell (GS-C2/PD-L1), the Luciferase reporter gene Luciferase expression regulated by the NFAT element in the effector cell is up-regulated. Using this principle, we inoculated (1) 4000 GS-C2/PD-L1 per well into 96-well plates for overnight culture; (2) the bispecific antibody MY2024/2084 and the positive control antibody Opdivo were diluted in a gradient (66.67nM, 22.22nM, 7.41nM, 2.47nM, 0.82nM, 0.27nM, 0.09nM, 0.03nM, 0.01nM) and the diluted antibodies were mixed with 1 × 10, respectively6Mixing GS-J2/PD-1 cells, adding into target cells at 37 deg.C with 5% CO2Culturing for 6 hours; (3) adding a Luciferase detection substrate, reacting for 5 minutes at room temperature, and then putting into an enzyme-labeling instrument for reading. The detection result is determined according to Normalized data (%) (RLU Test)samples-RLUBuffer control)/(RLUEC100 of Opdivo-RLUBuffer control) 100 processing.
The results are shown in fig. 8, EC50 of the control antibody Opdivo was 3.496, and EC50 of the bispecific antibody F was 3.085. It was thus demonstrated that the biological activity of the bispecific antibody F of the present application is similar to the activity of the marketed antibody Opdivo.
Example 8: CCK8 detection of proliferation inhibitory toxicity of antibody on A431 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) Pancreatin H292 cells, complete medium neutralization, PBS wash, according to 3X104Resuspended 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) Will be thinThe medium in the cells was aspirated and 100uL of diluted antibody was added. (5) After 72h, adding 10 uL/well of CCK8 solution, developing for 1-4h, and reading OD450 of each well by using a microplate reader after the color development is finished.
The experimental results shown in fig. 9 indicate that the inhibitory activity of the anti-PD-1/EGFR bispecific antibody F of the present application on cell proliferation is the same as that of cetuximab, which indicates that the bispecific antibody of the present application perfectly retains the biological activity of cetuximab.
Example 9: sample SEC analysis
The final selected double antibodies F were 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 BEH200SEC 3.5. mu. m7.8x300mm, the parameters are 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. 10, the double-antibody F only needs one-step affinity purification, and the purity of the double-antibody F can reach 98.05%.
The above examples show that the anti-PD-1/EGFR 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 98.05% after only one-step purification. The resulting bispecific antibody can bind EGFR-positive BxPC3 cells and simultaneously block the cell surface PD-1 interaction with PD-L1. In addition, the antibody has good affinity, not only has good biological activity of the PD-1 antibody, but also perfectly maintains the biological activity of the cetuximab. Therefore, the anti-PD-1/EGFR bispecific antibody disclosed 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-PD-1/EGFR bispecific antibody and application thereof
<130>P2018-1751
<150>CN201810936017.6
<151>2018-08-16
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580 585 590
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Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
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Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe Ser
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Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly
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Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Asp
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Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
690 695 700
Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val Pro
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Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Ala Ala IleGly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
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Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
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Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser
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Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly
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Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys
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Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg
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Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr
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Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
405 410 415
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
420 425 430
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
435 440 445
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
450 455 460
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
465 470 475 480
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
485 490 495
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
500 505 510
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
515 520 525
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
530 535 540
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
545 550 555 560
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
565 570 575
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
580 585 590
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
595 600 605
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
610 615 620
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
625 630 635 640
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
645 650 655
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
660 665 670
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
675 680 685
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
690 695 700
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
705 710 715 720
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
725 730 735
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
740 745 750
Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
755 760 765
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr
770 775 780
Tyr Leu Arg Phe Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly
785 790 795 800
Leu Glu Gly Val Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala
805 810 815
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn
820 825 830
Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
835 840 845
Tyr Tyr Cys Ala Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu
850 855 860
Ala Pro Leu Val Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
865 870 875 880
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val
885 890 895
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
900 905 910
Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe Ser Met
915 920 925
Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
930 935 940
Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg
945 950 955 960
Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Asp Met
965 970 975
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala
980 985 990
Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val Pro Tyr
995 1000 1005
Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1010 1015 1020
<210>16
<211>501
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>16
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
130 135 140
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
145 150 155 160
Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe Ser Met Gly Trp Phe Arg
165 170 175
Gln Val Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Gly Gly Asp
180 185 190
Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
195 200 205
Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg
210 215 220
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Val Leu Leu Asp
225 230 235 240
Gly Ser Phe Ser Leu Leu Ala Pro Leu Val Pro Tyr Lys Tyr Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
260 265 270
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
275 280 285
Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu
290 295 300
Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile
305 310 315 320
His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys
325 330 335
Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser
340 345 350
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu
355 360 365
Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr
370 375 380
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro
385 390 395 400
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
405 410 415
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
420 425 430
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
435 440 445
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
450 455 460
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
465 470 475 480
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
485 490 495
Asn Arg Gly Glu Cys
500
<210>17
<211>642
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>17
gacatcctgc tgacccagag ccccgtgatc ctgagcgtga gccccggcga gagggtgagc 60
ttcagctgca gggccagcca gagcatcggc accaacatcc actggtacca gcagaggacc 120
aacggcagcc ccaggctgct gatcaagtac gccagcgaga gcatcagcgg catccccagc 180
aggttcagcg gcagcggcag cggcaccgac ttcaccctga gcatcaacag cgtggagagc 240
gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac cttcggcgcc 300
ggcaccaagc tggagctgaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480
gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642
<210>18
<211>1347
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>18
caggtgcagc tgaagcagag cggccccggc ctggtgcagc ccagccagag cctgagcatc 60
acctgcaccg tgagcggctt cagcctgacc aactacggcg tgcactgggt gaggcagagc 120
cccggcaagg gcctggagtg gctgggcgtg atctggagcg gcggcaacac cgactacaac 180
acccccttca ccagcaggct gagcatcaac aaggacaaca gcaagagcca ggtgttcttc 240
aagatgaaca gcctgcagag caacgacacc gccatctact actgcgccag ggccctgacc 300
tactacgact acgagttcgc ctactggggc cagggcaccc tggtgaccgt gagcgccgcc 360
agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc 420
accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg 480
aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540
ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600
atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagagggt ggagcccaag 660
agctgcgaca agacccacac ctgccccccc tgccccgccc ccgaggccgc cggcggcccc 720
agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag gacccccgag 780
gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840
gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaacagc 900
acctacaggg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960
tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020
gccaagggcc agcccaggga gccccaggtg tacaccctgc cccccagcag ggaggagatg 1080
accaagaacc aggtgagcct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 1140
gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200
gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag caggtggcag 1260
cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320
aagagcctga gcctgagccc cggcaag 1347
<210>19
<211>2208
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>19
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcag cacctacctg aggttcagca tgggctggtt caggcaggtg 120
cccggcaagg gcctggaggg cgtggccgcc atcggcggcg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc aaggacaaca gcaagaacac cctgtacctg 240
gacatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgccgtgctg 300
ctggacggca gcttcagcct gctggccccc ctggtgccct acaagtacga ctactggggc 360
cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cagccaggtg 420
cagctgcagg agagcggcgg cggcctggtg cagcccggcg gcagcctgag gctgagctgc 480
gccgccagcg gcagcaccta cctgaggttc agcatgggct ggttcaggca ggtgcccggc 540
aagggcctgg agggcgtggc cgccatcggc ggcgacggca ggaccagcta cgccgacagc 600
gtgaagggca ggttcaccat cagcaaggac aacagcaaga acaccctgta cctggacatg 660
aacagcctga gggccgagga caccgccgtg tactactgcg ccgccgccgt gctgctggac 720
ggcagcttca gcctgctggc ccccctggtg ccctacaagt acgactactg gggccagggc 780
accctggtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 840
ggcagcggcg gcggcggcag ccaggtgcag ctgaagcaga gcggccccgg cctggtgcag 900
cccagccaga gcctgagcat cacctgcacc gtgagcggct tcagcctgac caactacggc 960
gtgcactggg tgaggcagag ccccggcaag ggcctggagt ggctgggcgt gatctggagc 1020
ggcggcaaca ccgactacaa cacccccttc accagcaggc tgagcatcaa caaggacaac 1080
agcaagagcc aggtgttctt caagatgaac agcctgcaga gcaacgacac cgccatctac 1140
tactgcgcca gggccctgac ctactacgac tacgagttcg cctactgggg ccagggcacc 1200
ctggtgaccg tgagcgccgc cagcaccaag ggccccagcg tgttccccct ggcccccagc 1260
agcaagagca ccagcggcgg caccgccgcc ctgggctgcc tggtgaagga ctacttcccc 1320
gagcccgtga ccgtgagctg gaacagcggc gccctgacca gcggcgtgca caccttcccc 1380
gccgtgctgc agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc 1440
agcctgggca cccagaccta catctgcaacgtgaaccaca agcccagcaa caccaaggtg 1500
gacaagaggg tggagcccaa gagctgcgac aagacccaca cctgcccccc ctgccccgcc 1560
cccgaggccg ccggcggccc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 1620
atgatcagca ggacccccga ggtgacctgc gtggtggtgg acgtgagcca cgaggacccc 1680
gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 1740
agggaggagc agtacaacag cacctacagg gtggtgagcg tgctgaccgt gctgcaccag 1800
gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcccgccccc 1860
atcgagaaga ccatcagcaa ggccaagggc cagcccaggg agccccaggt gtacaccctg 1920
ccccccagca gggaggagat gaccaagaac caggtgagcc tgacctgcct ggtgaagggc 1980
ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac 2040
aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc 2100
gtggacaaga gcaggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc 2160
ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaag 2208
<210>20
<211>2208
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>20
caggtgcagc tgaagcagag cggccccggc ctggtgcagc ccagccagag cctgagcatc 60
acctgcaccg tgagcggctt cagcctgacc aactacggcg tgcactgggt gaggcagagc 120
cccggcaagg gcctggagtg gctgggcgtg atctggagcg gcggcaacac cgactacaac 180
acccccttca ccagcaggct gagcatcaac aaggacaaca gcaagagcca ggtgttcttc 240
aagatgaaca gcctgcagag caacgacacc gccatctact actgcgccag ggccctgacc 300
tactacgact acgagttcgc ctactggggc cagggcaccc tggtgaccgt gagcgccgcc 360
agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc 420
accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg 480
aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540
ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600
atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagagggt ggagcccaag 660
agctgcgaca agacccacac ctgccccccc tgccccgccc ccgaggccgc cggcggcccc 720
agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag gacccccgag 780
gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840
gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaacagc 900
acctacaggg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960
tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020
gccaagggcc agcccaggga gccccaggtg tacaccctgc cccccagcag ggaggagatg 1080
accaagaacc aggtgagcct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 1140
gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200
gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag caggtggcag 1260
cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320
aagagcctga gcctgagccc cggcaagggc ggcggcggca gcggcggcgg cggcagcggc 1380
ggcggcggca gcggcggcgg cggcagccag gtgcagctgc aggagagcgg cggcggcctg 1440
gtgcagcccg gcggcagcct gaggctgagc tgcgccgcca gcggcagcac ctacctgagg 1500
ttcagcatgg gctggttcag gcaggtgccc ggcaagggcc tggagggcgt ggccgccatc 1560
ggcggcgacg gcaggaccag ctacgccgac agcgtgaagg gcaggttcac catcagcaag 1620
gacaacagca agaacaccct gtacctggac atgaacagcc tgagggccga ggacaccgcc 1680
gtgtactact gcgccgccgc cgtgctgctg gacggcagct tcagcctgct ggcccccctg 1740
gtgccctaca agtacgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 1800
ggcggcagcg gcggcggcag ccaggtgcag ctgcaggaga gcggcggcgg cctggtgcag 1860
cccggcggca gcctgaggct gagctgcgcc gccagcggca gcacctacct gaggttcagc 1920
atgggctggt tcaggcaggt gcccggcaag ggcctggagg gcgtggccgc catcggcggc 1980
gacggcagga ccagctacgc cgacagcgtg aagggcaggt tcaccatcag caaggacaac 2040
agcaagaaca ccctgtacct ggacatgaac agcctgaggg ccgaggacac cgccgtgtac 2100
tactgcgccg ccgccgtgct gctggacggc agcttcagcc tgctggcccc cctggtgccc 2160
tacaagtacg actactgggg ccagggcacc ctggtgaccg tgagcagc 2208
<210>21
<211>3069
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>21
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcag cacctacctg aggttcagca tgggctggtt caggcaggtg 120
cccggcaagg gcctggaggg cgtggccgcc atcggcggcg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc aaggacaaca gcaagaacac cctgtacctg 240
gacatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgccgtgctg 300
ctggacggca gcttcagcct gctggccccc ctggtgccct acaagtacga ctactggggc 360
cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cagccaggtg 420
cagctgcagg agagcggcgg cggcctggtg cagcccggcg gcagcctgag gctgagctgc 480
gccgccagcg gcagcaccta cctgaggttc agcatgggct ggttcaggca ggtgcccggc 540
aagggcctgg agggcgtggc cgccatcggc ggcgacggca ggaccagcta cgccgacagc 600
gtgaagggca ggttcaccat cagcaaggac aacagcaaga acaccctgta cctggacatg 660
aacagcctga gggccgagga caccgccgtg tactactgcg ccgccgccgt gctgctggac 720
ggcagcttca gcctgctggc ccccctggtg ccctacaagt acgactactg gggccagggc 780
accctggtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 840
ggcagcggcg gcggcggcag ccaggtgcag ctgaagcaga gcggccccgg cctggtgcag 900
cccagccaga gcctgagcat cacctgcacc gtgagcggct tcagcctgac caactacggc 960
gtgcactggg tgaggcagag ccccggcaag ggcctggagt ggctgggcgt gatctggagc 1020
ggcggcaaca ccgactacaa cacccccttc accagcaggc tgagcatcaa caaggacaac 1080
agcaagagcc aggtgttctt caagatgaac agcctgcaga gcaacgacac cgccatctac 1140
tactgcgcca gggccctgac ctactacgac tacgagttcg cctactgggg ccagggcacc 1200
ctggtgaccg tgagcgccgc cagcaccaag ggccccagcg tgttccccct ggcccccagc 1260
agcaagagca ccagcggcgg caccgccgcc ctgggctgcc tggtgaagga ctacttcccc 1320
gagcccgtga ccgtgagctg gaacagcggc gccctgacca gcggcgtgca caccttcccc 1380
gccgtgctgc agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc 1440
agcctgggca cccagaccta catctgcaac gtgaaccaca agcccagcaa caccaaggtg 1500
gacaagaggg tggagcccaa gagctgcgac aagacccaca cctgcccccc ctgccccgcc 1560
cccgaggccg ccggcggccc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 1620
atgatcagca ggacccccga ggtgacctgc gtggtggtgg acgtgagcca cgaggacccc 1680
gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 1740
agggaggagc agtacaacag cacctacagg gtggtgagcg tgctgaccgt gctgcaccag 1800
gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcccgccccc 1860
atcgagaaga ccatcagcaa ggccaagggc cagcccaggg agccccaggt gtacaccctg 1920
ccccccagca gggaggagat gaccaagaac caggtgagcc tgacctgcct ggtgaagggc 1980
ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac 2040
aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc 2100
gtggacaaga gcaggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc 2160
ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaaggg cggcggcggc 2220
agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcca ggtgcagctg 2280
caggagagcg gcggcggcct ggtgcagccc ggcggcagcc tgaggctgag ctgcgccgcc 2340
agcggcagca cctacctgag gttcagcatg ggctggttca ggcaggtgcc cggcaagggc 2400
ctggagggcg tggccgccat cggcggcgac ggcaggacca gctacgccga cagcgtgaag 2460
ggcaggttca ccatcagcaa ggacaacagc aagaacaccc tgtacctgga catgaacagc 2520
ctgagggccg aggacaccgc cgtgtactac tgcgccgccg ccgtgctgct ggacggcagc 2580
ttcagcctgc tggcccccct ggtgccctac aagtacgact actggggcca gggcaccctg 2640
gtgaccgtga gcagcggcgg cggcggcagc ggcggcggca gccaggtgca gctgcaggag 2700
agcggcggcg gcctggtgca gcccggcggc agcctgaggc tgagctgcgc cgccagcggc 2760
agcacctacc tgaggttcag catgggctgg ttcaggcagg tgcccggcaa gggcctggag 2820
ggcgtggccg ccatcggcgg cgacggcagg accagctacg ccgacagcgt gaagggcagg 2880
ttcaccatca gcaaggacaa cagcaagaac accctgtacc tggacatgaa cagcctgagg 2940
gccgaggaca ccgccgtgta ctactgcgcc gccgccgtgc tgctggacgg cagcttcagc 3000
ctgctggccc ccctggtgcc ctacaagtac gactactggg gccagggcac cctggtgacc 3060
gtgagcagc 3069
<210>22
<211>1503
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>22
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcag cacctacctg aggttcagca tgggctggtt caggcaggtg 120
cccggcaagg gcctggaggg cgtggccgcc atcggcggcg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc aaggacaaca gcaagaacac cctgtacctg 240
gacatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgccgtgctg 300
ctggacggca gcttcagcct gctggccccc ctggtgccct acaagtacga ctactggggc 360
cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cagccaggtg 420
cagctgcagg agagcggcgg cggcctggtg cagcccggcg gcagcctgag gctgagctgc 480
gccgccagcg gcagcaccta cctgaggttc agcatgggct ggttcaggca ggtgcccggc 540
aagggcctgg agggcgtggc cgccatcggc ggcgacggca ggaccagcta cgccgacagc 600
gtgaagggca ggttcaccat cagcaaggac aacagcaaga acaccctgta cctggacatg 660
aacagcctga gggccgagga caccgccgtg tactactgcg ccgccgccgt gctgctggac 720
ggcagcttca gcctgctggc ccccctggtg ccctacaagt acgactactg gggccagggc 780
accctggtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 840
ggcagcggcg gcggcggcag cgacatcctg ctgacccaga gccccgtgat cctgagcgtg 900
agccccggcg agagggtgag cttcagctgc agggccagcc agagcatcgg caccaacatc 960
cactggtacc agcagaggac caacggcagc cccaggctgc tgatcaagta cgccagcgag 1020
agcatcagcg gcatccccag caggttcagc ggcagcggca gcggcaccga cttcaccctg 1080
agcatcaaca gcgtggagag cgaggacatc gccgactact actgccagca gaacaacaac 1140
tggcccacca ccttcggcgc cggcaccaag ctggagctga agaggaccgt ggccgccccc 1200
agcgtgttca tcttcccccc cagcgacgag cagctgaaga gcggcaccgc cagcgtggtg 1260
tgcctgctga acaacttcta ccccagggag gccaaggtgc agtggaaggt ggacaacgcc 1320
ctgcagagcg gcaacagcca ggagagcgtg accgagcagg acagcaagga cagcacctac 1380
agcctgagca gcaccctgac cctgagcaag gccgactacg agaagcacaa ggtgtacgcc 1440
tgcgaggtga cccaccaggg cctgagcagc cccgtgacca agagcttcaa caggggcgag 1500
tgc 1503
<210>23
<211>6
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>23
Gln Ser Ile Gly Thr Asn
1 5
<210>24
<211>3
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>24
Tyr Ala Ser
1
<210>25
<211>9
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>25
Gln Gln Asn AsnAsn Trp Pro Thr Thr
1 5
<210>26
<211>8
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>26
Gly Phe Ser Leu Thr Asn Tyr Gly
1 5
<210>27
<211>7
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>27
Ile Trp Ser Gly Gly Asn Thr
1 5
<210>28
<211>13
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>28
Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr
1 5 10
<210>29
<211>107
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>29
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210>30
<211>119
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>30
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 4045
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210>31
<211>267
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>31
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 5560
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
130 135 140
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
145 150 155 160
Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe Ser Met Gly Trp Phe Arg
165 170 175
Gln Val Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Gly Gly Asp
180 185 190
Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
195 200 205
Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg
210 215220
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Val Leu Leu Asp
225 230 235 240
Gly Ser Phe Ser Leu Leu Ala Pro Leu Val Pro Tyr Lys Tyr Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
260 265

Claims (10)

1. A bifunctional antibody, wherein said bifunctional antibody comprises:
(a) antibodies against Epidermal Growth Factor (EGFR); and
(b) a bivalent form of a nanobody against PD-1 linked to the anti-Epidermal Growth Factor (EGFR) 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 FDA0001803880610000011
wherein,
each D is independently a nanobody against PD-1 or no, and at least one D is a nanobody against PD-1;
l1, L2, L3, L4, L5, L6 are each independently a bond or a linker element;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
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 FDA0001803880610000012
wherein,
each D represents a nano antibody against PD-1;
l1, L2, L3, L4 represent no or linker elements, respectively;
VL represents the light chain variable region of an anti-EGFR antibody;
CL represents the light chain constant region of an anti-EGFR antibody;
VH represents the heavy chain variable region of an anti-EGFR antibody;
CH represents the heavy chain constant region of an anti-EGFR antibody;
"-" represents a disulfide bond;
"-" represents a peptide bond;
wherein the bifunctional antibody has the activity of simultaneously binding to EGFR and binding to PD-1.
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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CN114106190A (en) * 2020-08-31 2022-03-01 普米斯生物技术(珠海)有限公司 anti-VEGF/PD-L1 bispecific antibody and application thereof
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CN114159557A (en) * 2021-11-09 2022-03-11 北京东方百泰生物科技股份有限公司 Combined medicine composition for treating tumor diseases and application thereof
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WO2024222662A1 (en) * 2023-04-23 2024-10-31 石药集团巨石生物制药有限公司 Use of antibody drug conjugate
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