CN112745392A - anti-PD-L1/CD 47 bispecific antibody and application thereof - Google Patents

anti-PD-L1/CD 47 bispecific antibody and application thereof Download PDF

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CN112745392A
CN112745392A CN201911046449.0A CN201911046449A CN112745392A CN 112745392 A CN112745392 A CN 112745392A CN 201911046449 A CN201911046449 A CN 201911046449A CN 112745392 A CN112745392 A CN 112745392A
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CN112745392B (en
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万亚坤
朱敏
盖军伟
沈晓宁
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Shanghai Luoqi Biomedical Technology Co ltd
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The invention provides anti-PD-L1/CD 47 bispecific antibodies and uses thereof. In particular, the invention provides a bispecific antibody comprising (a) a PD-L1 single domain antibody and (b) a CD47 single domain antibody. The present invention provides a coding sequence encoding the bispecific antibody, a corresponding expression vector and a host cell capable of expressing the bispecific antibody, and a method for producing the bispecific antibody of the present invention. The bispecific antibody can simultaneously target PD-L1 and CD47, can remarkably activate T cells, can effectively promote phagocytosis of Jurkat cells by macrophages, does not cause human erythrocyte agglutination, and has good application prospect.

Description

anti-PD-L1/CD 47 bispecific antibody and application thereof
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, and more particularly relates to an anti-PD-L1/CD 47 bispecific antibody and application thereof.
Background
The tumor immune response of the body is the result of a combination of the immune function status of the host and the immunogenicity of the tumor. Tumor cells can cause immune escape of tumor cells by abnormally expressing immunosuppressive molecules. Recent studies show that the PD-1/PD-L1 inhibition pathway plays an important role in tumor immune response, mediation of transplant rejection, occurrence and development of autoimmune diseases and chronic viral infection. PD-L1 is expressed on the surface of activated T cells, B cells, macrophages, dendritic cells, tumor cells, etc., or immune-shielded tissues (placenta, vascular endothelium, etc.). The targeting PD-1/PD-L1 antibody promotes the proliferation and cytokine release of T cells in vitro by blocking T cell inhibitory signaling pathways.
The CD47 molecule is a transmembrane glycoprotein widely expressed on the surface of various cells, and plays an important role in immune response, tumor immunity, and the like by binding to its receptor integrin ligand, signal-regulatory protein (sirpa), thrombin-sensitive protein (TSP), and the like. In recent years, the CD47/SIRPa signal channel mediated ' Don't eat me ' inhibits the phagocytic activity of macrophages, and is closely related to the occurrence of various malignant solid tumors and blood tumors. However, CD47 is not strictly a tumor tissue specific antigen, and normal tissue cells, such as red blood cells, also express CD 47. The anti-CD 47 monoclonal antibody can cause the phenomenon of 'antigen sinking' due to the expression of a large amount of CD47 on the cell surface of erythrocytes and the like after being administrated in vivo, and even cause serious blood toxicity due to off-target effect.
The tumor cells transmit a ' Don't find me ' signal through high-expression PD-L1 molecules, and the failure or the apoptosis of effector CTL cells is induced; the ' Don't eat me ' signal is transmitted by a high-expression CD47 molecule, so that phagocytosis of macrophages is avoided. Antibodies targeting CD47 can initiate T cell antigen presentation, induce T cell immune function, and are functionally complementary to anti-PD-1/PD-L1. Research shows that the mouse has the synergistic effect of inhibiting tumor by blocking CD47 and PD-L1 signal channels simultaneously, and can activate innate immunity and adaptive immunity simultaneously and promote the innate immunity and the adaptive immunity mutually, so that the autoimmune system of the body is utilized to inhibit tumor growth to the maximum extent. Chinese patent CN107459578A discloses a bifunctional fusion protein targeting CD47 and PD-L1, Chinese patent CN109970860A discloses an anti-CD 47/PD-L1 bispecific antibody, Taiwan patent TW201922781A discloses an antibody specific to CD47 and PD-L1, and PCT application WO2019080883A1 discloses a recombinant fusion protein capable of simultaneously combining CD47, PD-L1 and FcR.
At present, no bispecific single-domain antibody targeting PD-L1/CD47 is disclosed in the market, and the single-domain antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like as a new generation of antibody diagnosis and treatment, and plays an beyond-imaginable huge function in immune experiments, diagnosis and treatment. Therefore, the development of the bispecific single domain antibody which simultaneously targets PD-L1 and CD47 has potential market value and clinical application value.
Disclosure of Invention
The invention aims to provide an anti-PD-L1/CD 47 bispecific antibody and application thereof.
In a first aspect of the invention, there is provided a bispecific antibody comprising: anti-PD-L1 single domain antibodies and anti-CD 47 single domain antibodies.
In another preferred embodiment, the bispecific antibody comprises 1-4 anti-PD-L1 single domain antibodies, preferably 2 anti-PD-L1 single domain antibodies, more preferably the two anti-PD-L1 single domain antibodies form an anti-PD-L1 single domain antibody dimer.
In another preferred embodiment, the bispecific antibody comprises 1-4 anti-CD 47 single domain antibodies, preferably 2 anti-CD 47 single domain antibodies, more preferably the two anti-CD 47 single domain antibodies form an anti-CD 47 single domain antibody dimer.
In another preferred embodiment, the bispecific antibody further comprises an Fc fragment, preferably the Fc fragment comprises a CH2 domain and a CH3 domain.
In another preferred embodiment, the Fc fragment is an IgG4 type Fc fragment.
In another preferred embodiment, the bispecific antibody has a structure represented by formula i (a) or i (b) from N-terminus to C-terminus:
P-L1-P-L2-Fc-L3-B-L4-B formula I (a)
B-L1-B-L2-Fc-L3-P-L4-P formula I (B)
Wherein the content of the first and second substances,
"-" is a peptide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is an anti-PD-L1 single domain antibody,
b is an anti-CD 47 single domain antibody, and
fc is the Fc segment of the antibody.
In another preferred embodiment, the bispecific antibody has a structure represented by formula i (a) from N-terminus to C-terminus.
In another preferred embodiment, the amino acid sequence of the anti-PD-L1 single-domain antibody is shown as SEQ ID No. 1, and the nucleotide sequence is shown as SEQ ID No. 8.
In another preferred embodiment, the PD-L1 is human PD-L1.
In another preferred embodiment, the anti-PD-L1 single domain antibody can block the interaction between PD-1 and PD-L1.
In another preferred embodiment, the amino acid sequence of the anti-CD 47 single domain antibody is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 10.
In another preferred embodiment, the CD47 is human CD 47.
In another preferred embodiment, the anti-CD 47 single domain antibody can block the interaction between CD47 and sirpa.
In another preferred embodiment, said L1, L3, and L4 are linker elements.
In another preferred embodiment, the sequence of the linker element is (4GS) n, wherein n is a positive integer (e.g. 1, 2, 3, 4, 5 or 6), preferably n ═ 4.
In another preferred embodiment, the amino acid sequence of the linker element is as shown in SEQ ID No. 2 and the nucleotide sequence is as shown in SEQ ID No. 9.
In another preferred embodiment, said L2 is a peptide bond.
In another preferred embodiment, the Fc segment is shown as SEQ ID NO. 6 at position 273 and 501.
In another preferred embodiment, the amino acid sequence of the bispecific antibody is shown in SEQ ID No. 6.
In a second aspect of the invention, there is provided a bispecific fusion protein which is a dimer formed from two bispecific antibodies according to the first aspect of the invention.
In another preferred embodiment, the bispecific fusion protein comprises 4 anti-PD-L1 single domain antibodies and 4 CD47 single domain antibodies.
In another preferred embodiment, the bispecific fusion protein forms a dimer through the Fc segment.
In another preferred embodiment, the dimer includes a homodimer and a heterodimer.
In another preferred embodiment, the bispecific fusion protein has a structure represented by formula II from N-terminus to C-terminus:
Figure BDA0002254258610000031
wherein the content of the first and second substances,
"-" is a peptide bond, "║" is a disulfide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is an anti-PD-L1 single domain antibody,
b is an anti-CD 47 single domain antibody, and
fc is the Fc segment of the antibody.
In a third aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the bispecific antibody of the first aspect of the invention, the bispecific fusion protein of the second aspect of the invention.
In another preferred embodiment, the polynucleotide encodes the bispecific antibody of the first aspect of the invention and the polynucleotide is as set forth in SEQ ID No. 13.
In another preferred embodiment, the polynucleotide comprises DNA or RNA.
In a fourth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
In a fifth aspect of the invention, there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having a polynucleotide according to the third aspect of the invention integrated into its genome;
alternatively, the host cell expresses the bispecific antibody of the first aspect of the invention or the bispecific fusion protein of the second aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: escherichia coli, yeast cells, mammalian cells.
In a sixth aspect of the invention, there is provided a method of producing a bispecific antibody comprising the steps of:
(a) culturing the host cell of the fifth aspect of the invention under suitable conditions, thereby obtaining a culture comprising the bispecific antibody; and
(b) purifying and/or isolating the culture obtained in step (a) to obtain said bispecific antibody.
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 seventh aspect of the invention, there is provided an immunoconjugate comprising:
(a) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second 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, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein or VLP, or a combination thereof.
In another preferred embodiment, the radionuclide includes:
(i) a diagnostic isotope selected from the group consisting of: tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indobenzodiazepines (indoxazepines) and benzodiazepines (oxyphenoxazepines)), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoid, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthrax toxin dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a chain, modeccin a chain, alpha-sarcina, gelonin, mitogelonin (mitogellin), restrictocin (rettstricon), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.
In another preferred embodiment, the conjugated moiety is a detectable label.
In another preferred embodiment, the conjugate 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), 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).
In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g. bivalent) bispecific fusion protein according to the second aspect of the invention.
In another preferred embodiment, said multivalent is a bispecific fusion protein according to the second aspect of the invention comprising multiple repeats in the amino acid sequence of said immunoconjugate.
In an eighth aspect of the invention, there is provided the use of a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, for the preparation of (a) an agent for the detection of a PD-L1 and/or CD47 molecule; (b) a Chinese medicinal composition for preventing and treating tumor.
In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.
In another preferred embodiment, the agent is one or more agents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particles and imaging agent.
In another preferred example, the reagent for detecting PD-L1 and/or CD47 molecules is a contrast agent for (in vivo) detecting PD-L1 and/or CD47 molecules.
In another preferred embodiment, the assay is an in vivo assay or an in vitro assay.
In another preferred embodiment, the detection comprises flow detection and cell immunofluorescence detection.
In another preferred embodiment, the medicament is used for blocking the interaction of PD-1 and PD-L1 and simultaneously blocking the interaction of CD47 and SIRPa.
In another preferred embodiment, the tumor includes, but is not limited to, acute myelocytic leukemia, chronic myelocytic leukemia, multiple myelopathy, non-hodgkin's lymphoma, colorectal cancer, breast cancer, large intestine cancer, stomach cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymph cancer, adrenal tumor, or bladder tumor.
In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention; and (ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the conjugation moiety of the immunoconjugate is a drug, toxin, and/or therapeutic isotope.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.
In another preferred embodiment, the other drug for treating tumor comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, or pembrolizumab.
In another preferred embodiment, the pharmaceutical composition is used for blocking the interaction of PD-1 and PD-L1 and simultaneously blocking the interaction of CD47 and SIRPa.
In another preferred embodiment, the pharmaceutical composition is used for blocking a PD-1/PD-L1 signal pathway.
In another preferred embodiment, the pharmaceutical composition is used for treating tumors expressing PD-L1 protein (i.e., PD-L1 positive).
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for preventing and treating tumors.
In a tenth aspect of the invention, there is provided the use of one or more of the bispecific antibody of the first aspect of the invention, the bispecific fusion protein of the second aspect of the invention selected from the group consisting of: (i) for detecting human PD-L1 molecules and/or CD47 molecules; (ii) for streaming detection; (iii) for cellular immunofluorescence detection; (iv) for the treatment of tumors; (v) for use in tumor diagnosis; (vi) for blocking the interaction of PD-1 and PD-L1; and (vii) for blocking the interaction of CD47 and sirpa.
In another preferred embodiment, the tumor is a tumor expressing PD-L1 protein (i.e., PD-L1 positive).
In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
In another preferred embodiment, the antibody is an antibody against PD-L1 and/or CD 47.
In an eleventh aspect of the present invention, there is provided a recombinant protein having: (i) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention; and (ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag, an HA tag and an Fc tag.
In another preferred embodiment, the recombinant protein specifically binds to PD-L1 and/or CD 47.
In a twelfth aspect of the invention there is provided the use of a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, for the preparation of a medicament, a reagent, a detection plate or a kit; wherein the reagent, assay plate or kit is for: detecting PD-L1 and/or CD47 in the sample; wherein the medicament is used for treating or preventing a tumor expressing PD-L1 (i.e., PD-L1 positive) or a tumor expressing CD 47.
In a thirteenth aspect of the invention, there is provided a method of detecting PD-L1 and/or CD47 in a sample, the method comprising the steps of: (1) contacting a sample with a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention; (2) detecting the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of PD-L1 and/or CD47 in the sample.
In a fourteenth aspect of the invention, there is provided a method of treating a disease, the method comprising administering to a subject in need thereof a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention or an immunoconjugate according to the seventh aspect of the invention.
In another preferred embodiment, the subject comprises a mammal, such as a human.
In a fifteenth aspect of the invention there is provided a PD-L1 and/or CD47 detection reagent comprising an immunoconjugate according to the seventh aspect of the invention and a detectably acceptable carrier.
In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.
In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert, aqueous carrier medium.
In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particles and imaging agent.
In another preferred embodiment, the detection reagent is used for in vivo detection.
In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as water solution, injection, freeze-dried powder, tablet, buccal agent and aerosol).
In a sixteenth aspect of the invention, there is provided a kit for detecting PD-L1 and/or CD47, the kit comprising an immunoconjugate according to the seventh aspect of the invention or a detection reagent according to the fifteenth aspect of the invention, and instructions.
In another preferred embodiment, the instructions describe that the kit is used for non-invasively detecting the expression of PD-L1 and/or CD47 in a subject to be detected.
In another preferred embodiment, the kit is used for detecting tumors expressing PD-L1 protein (i.e., PD-L1 positive).
In a seventeenth aspect of the invention, there is provided a use of an immunoconjugate of the seventh aspect of the invention for the preparation of a contrast agent for the in vivo detection of PD-L1 protein and/or CD47 protein.
In another preferred embodiment, the detection is used for the diagnosis or prognosis of cancer.
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. 1A shows a schematic structural diagram of an anti-PD-L1/CD 47 bispecific antibody A (diabody A).
FIG. 1B shows a schematic structural diagram of anti-PD-L1/CD 47 bispecific antibody B (diabody B).
FIG. 1C shows a schematic structural diagram of anti-PD-L1/CD 47 bispecific antibody C (diabody C).
FIG. 1D shows a schematic structural diagram of anti-PD-L1/CD 47 bispecific antibody D (diabody D).
FIG. 2 shows the result of the detection of the blocking activity of the anti-PD-L1/CD 47 bispecific antibody on the CD47/SIRPa pathway. Double antibody C (IC)506.226nM) and diabody D (IC)504.907nM) retained better activity of blocking the CD47/SIRPa interaction, with better activity of the diabody D.
FIG. 3 shows the result of detection of the blocking activity of the anti-PD-L1/CD 47 bispecific antibody against the PD-1/PD-L1 pathway. The blocking activity of the double anti-C is closer to that of the control antibody, namely double anti-C (IC)506.397nM) has better blocking activity on PD-1/PD-L1 than that of double-antibody D (IC)50＝10.04nM)。
FIGS. 4A-4D show the results of affinity assays for anti-PD-L1/CD 47 bispecific antibodies, respectively. Specifically, FIG. 4A shows the affinity assay for the control antibody CD47 Nb-Fc, with an affinity of 6.13E-09M. FIG. 4B shows the result of affinity assay of the diabody C-CD47, the affinity is 6.15E-09M. The results in FIG. 4A and FIG. 4B show that the affinity of the dual anti-C-CD 47 is consistent with that of the control antibody CD47 Nb-Fc. FIG. 4C shows the affinity assay result for the control antibody PD-L1 Nb-Fc, with an affinity of 2.65E-08M. FIG. 4D shows the result of affinity assay of the diabody C-PD-L1, the affinity is 2.66E-08M. The results in FIG. 4C and FIG. 4D show that the affinity of the dual anti-C-PD-L1 is also consistent with that of the control antibody PD-L1 Nb-Fc.
FIG. 5A shows the results of the activity identification of anti-PD-L1/CD 47 bispecific antibody coated with CD47 antigen while binding to dual targets. FIG. 5B shows the results of the activity identification of anti-PD-L1/CD 47 bispecific antibody coated with PD-L1 antigen while binding to dual targets. The results of FIG. 5A and FIG. 5B show that the anti-PD-L1/CD 47 bispecific antibody C of the present invention can bind to both CD47 and PD-L1.
FIGS. 6A and 6B show the results of functional assays for T cell activation by anti-PD-L1/CD 47 bispecific antibodies in Donor1 and Donor2, respectively. The result shows that the content of IL-2 in the experimental group and the control group is obviously higher than that in the negative control group, and the candidate double-anti-C and the control antibody both show obvious activation effect on T cells.
FIG. 7 shows the results of the anti-PD-L1/CD 47 bispecific antibody induced phagocytosis of tumor cells by macrophages. Both the control antibody and the candidate diabody C are effective in promoting phagocytosis of Jurkat cells by macrophages.
FIGS. 8A and 8B show the results of agglutination tests of human erythrocytes by the anti-PD-L1/CD 47 bispecific antibody in Donor1 and Donor2, respectively. The results indicate that the control antibody Hu5F9 can obviously cause human erythrocyte agglutination, the control antibody B6H12 can cause slight erythrocyte agglutination reaction at lower antibody concentration, and the CD47 single-domain antibody and the candidate double-antibody C can not cause human erythrocyte agglutination.
FIGS. 9A and 9B show the results of stability assays for anti-PD-L1/CD 47 bispecific antibodies at 4 ℃ and 25 ℃, respectively. The result shows that the double-resistant C is stable under the condition of 4 ℃; the di-anti-C is partially degraded under accelerated conditions of 25 ℃.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly obtained an anti-PD-L1/CD 47 bispecific antibody comprising an anti-PD-L1 single domain antibody and an anti-CD 47 single domain antibody. Experiments show that the bispecific antibody has better binding activity to PD-L1 and CD47 molecules, can block the interaction between PD-1 and PD-L1 and the interaction between CD47 and SIRPa, and has good antitumor activity. The present invention has been completed based on this finding.
The bispecific antibodies of the invention can form dimers, i.e., 8-valent fusion proteins comprising 4 anti-PD-L1 single domain antibodies and 4 anti-CD 47 single domain antibodies, through an Fc fragment. Applicants found that the 8-valent fusion protein of the invention has superior blocking activity for the CD47/SIRPa pathway compared to a 4-valent fusion protein comprising 2 anti-PD-L1 single domain antibodies and 2 anti-CD 47 single domain antibodies.
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
As used herein, the terms "bispecific antibody of the invention", "diabody of the invention", "anti-PD-L1/CD 47 bispecific antibody" have the same meaning and all refer to bispecific antibodies that specifically recognize and bind to PD-L1 and CD 47.
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. 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 VHH", "nanobody" have the same meaning, referring to the variable region of a cloned antibody heavy chain, constructing a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Nanobodies (VHHs) consisting of only one heavy chain variable region are typically constructed by first obtaining an antibody that naturally lacks light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partially-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
As used herein, the term "framework region" (FR) refers to amino acid sequences inserted between CDRs, i.e., those portions of the light and heavy chain variable regions of an immunoglobulin that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR 4-H. Accordingly, the light chain variable domain may thus be referred to as (FR1-L) - (CDR1-L) - (FR2-L) - (CDR2-L) - (FR3-L) - (CDR3-L) - (FR4-L) and the heavy chain variable domain may thus be referred to as (FR1-H) - (CDR1-H) - (FR2-H) - (CDR2-H) - (FR3-H) - (CDR3-H) - (FR 4-H). Preferably, the FRs of the present invention are human antibody FRs or derivatives thereof that are substantially identical, i.e., 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, to the FRs of a naturally occurring human antibody.
Knowing the amino acid sequences of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR 4-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework regions of a naturally occurring human antibody.
As used herein, the term "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.
As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention to form conjugates. The invention also includes a cell surface marker or antigen that binds to the PD-L1/CD47 bispecific antibody or fragment thereof.
As used herein, the terms "heavy chain variable region" and "VH"may be used interchangeably.
As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and all refer to a polypeptide that specifically binds to PD-L1 and/or CD47 protein, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain the initial methionine.
The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy chain variable region, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with CDRs whose homology to the CDRs identified herein is greater than 90% (preferably greater than 95%, most preferably greater than 98%).
The invention includes not only intact 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 antibody of the present invention refers to a double antibody having binding activity to PD-L1 and/or CD47 proteins. The term also includes variants of polypeptides having the same function as the antibodies of the invention, comprising the same CDR regions. 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 antibodies of the invention.
Variants of the polypeptide 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.
The invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the single domain antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody 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
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
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 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 antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations 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. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
Bispecific antibodies
The invention provides an anti-PD-L1/CD 47 bispecific antibody comprising: anti-PD-L1 single domain antibodies and anti-CD 47 single domain antibodies.
In a preferred embodiment, the bispecific antibody comprises two anti-PD-L1 single domain antibodies and two anti-CD 47 single domain antibodies, and the bispecific antibody can form a dimer, i.e., a bispecific fusion protein comprising 4 anti-PD-L1 single domain antibodies and 4 anti-CD 47 single domain antibodies, as shown in figure 1C.
In fact, based on the anti-CD 47 single domain antibody and the anti-PD-L1 single domain antibody of the present invention, applicants constructed bispecific antibodies of various structures in the construction process of bispecific antibodies, including 1 anti-PD-L1 single domain antibody and 1 anti-CD 47 single domain antibody in addition to 2 anti-PD-L1 single domain antibodies and 2 anti-CD 47 single domain antibodies. The structure of the dimeric fusion protein formed by 2 anti-PD-L1 single-domain antibodies and 2 anti-CD 47 single-domain antibodies is shown in FIGS. 1C and 1D. A dimeric fusion protein formed by bispecific antibodies comprising 1 anti-PD-L1 single domain antibody and 1 anti-CD 47 single domain antibody, the structure of which is shown in fig. 1A and 1B. The experimental results show that the fusion protein shown in figure 1C and figure 1D has better blocking activity to CD47/SIRPa pathway compared with the fusion protein with the structure shown in figure 1A and figure 1B; whereas the fusion protein shown in FIG. 1D has better blocking activity in the PD-1/PD-L1 pathway than the fusion protein shown in FIG. 1C.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the invention can be directly used for binding PD-L1 and/or CD47 protein molecules, and thus can be used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single domain antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Labeled single domain antibodies
In a preferred embodiment of the invention, the single domain antibody carries a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, coloured labels or fluorescent labels.
The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the invention, the PD-L1/CD47 bispecific antibody may be labeled with colloidal gold, resulting in a colloidal gold-labeled single domain antibody.
The PD-L1/CD47 bispecific antibody has good specificity.
Detection method
The invention also relates to a method for detecting PD-L1 and/or CD47 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of PD-L1 and/or CD47 protein in the solubilized sample.
The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.
Reagent kit
The present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
The invention also provides a detection kit for detecting the level of PD-L1 and/or CD47, which comprises an antibody for identifying PD-L1 and/or CD47 protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.
Applications of
As described above, the single domain antibody of the present invention has wide biological and clinical applications, and its applications relate to many fields such as diagnosis and treatment of diseases related to PD-L1 and/or CD47, research in basic medicine, and biological research. A preferred application is for clinical diagnosis and targeted therapy against PD-L1 and/or CD47, such as tumor therapy.
The main advantages of the invention include:
(a) the bispecific antibodies of the invention can bind to both CD47 and PD-L1.
(b) The bispecific antibody of the invention can obviously activate T cells and effectively promote phagocytosis of Jurkat cells by macrophages.
(c) The bispecific antibody of the present invention does not cause agglutination of human erythrocytes.
(d) The bispecific antibodies of the invention are storage stable at non-formulation conditions at 4 ℃.
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 by weight.
Example 1: molecular structure design and expression of PD-L1/CD47 bispecific antibody
The inventors designed bispecific antibodies that bind both human PD-L1 and CD47 extracellular domain. Wherein, the PD-L1 single-domain antibody sequence is derived from Chinese patent CN201810151835.5, and the CD47 single-domain antibody sequence is derived from Chinese patent CN 201810151752.6.
While various structures of the PD-L1/CD47 bispecific antibody molecule were designed and tested for activity, this example exemplifies only four structures of the diabody molecule, namely, diabody A as shown in FIG. 1A, diabody B as shown in FIG. 1B, diabody C as shown in FIG. 1C, and diabody D as shown in FIG. 1D, wherein diabody A and diabody B are in the form of tetravalent antibody, and diabody C and diabody D are in the form of octahedral antibody.
Wherein, the amino acid sequence of the double-antibody A is SEQ ID NO. 4, and the nucleotide sequence is SEQ ID NO. 11; the amino acid sequence of the double antibody B is SEQ ID NO. 5, and the nucleotide sequence is SEQ ID NO. 12; the amino acid sequence of the double-antibody C is SEQ ID NO. 6, and the nucleotide sequence is SEQ ID NO. 13; the amino acid sequence of the double-antibody D is SEQ ID NO. 7, and the nucleotide sequence is SEQ ID NO. 14.
Specifically, the N end of the double-antibody A is a bivalent PD-L1 single-domain antibody, the C end is a bivalent CD47 single-domain antibody, and the bivalent PD-L1 single-domain antibody and the bivalent CD47 single-domain antibody are connected through a human Fc fragment and a flexible linker; the double-antibody B is provided with a bivalent CD47 single-domain antibody at the N end and a bivalent PD-L1 single-domain antibody at the C end, and the bivalent CD47 single-domain antibody and the bivalent PD-L1 single-domain antibody are connected through a human Fc fragment and a flexible linker; the N end of the double-antibody C is a tetravalent PD-L1 single-domain antibody, the C end is a tetravalent CD47 single-domain antibody, and the double-antibody C and the terminal are connected through a flexible linker and a human Fc fragment; the N terminal of the double-antibody D is a tetravalent CD47 single-domain antibody, and the N terminal is a tetravalent PD-L1 single-domain antibody which are connected through a flexible linker and a human Fc fragment.
The amino acids of the antibody are optimized into a base sequence according to CHO species codons and then cloned between EcoRV and PacI enzyme cutting sites on a pCHO1.0 vector, then the cloned plasmid and an equivalent amount MAX are mixed to transfect CHO-S cells for antibody expression, and the expressed supernatant is combined with Protein A to obtain the purified multispecific antibody.
Example 2: comparison of blocking Activity of PD-L1/CD47 bispecific antibody against CD47/SIRPa pathway
(1) Taking 5X 10 samples of each5Jurkat cells (highly expressing human CD47) were loaded with PD-L1/CD47 bispecific antibody and positive control CD47 single domain antibody Fc fusion protein (Nb-Fc) in 0.5% BSA-PBS buffer at a dilution gradient of 514nM, 257nM, 129nM, 64.3nM, 32.1nM, 16.1nM, 8.03nM, 4.02nM, 2.00nM, 1.00nM, 0.50nM, 0.25nM, 100uL per sample, 5ug hSIRPa (ECD) -Fc-Biotin added simultaneously to all samples, and incubated at 4 ℃ for 20 min; (2) the cells were washed 2 times with PBS, SA-PE from eBioscience was added, incubated at 4 ℃ for 20min, and the cells were washed 2 times with PBS and then detected with a flow cytometer (BD FACS Calibur) and processed with the graphpad prism 6 software.
The results are shown in FIG. 2: blocking activity of the octavalent antibody is superior to that of the tetravalent antibody, tetravalent antibody A (IC)5010.751nM) and B (IC)508.302nM) compared to the original CD47 single domain antibody (IC)504.487nM), and double antibody C (IC)506.226nM) and diabody D (IC)504.907nM) retained better activity of blocking the CD47/SIRPa interaction, with better activity of the diabody D.
Example 3: comparison of blocking Activity of PD-L1/CD47 bispecific antibody against PD-1/PD-L1 pathway
Taking 3X 10 samples of each5A375/PD-L1 cells were stably transfected into 0.5% BSA-PBS buffer, and a test double-antibody and control antibody PD-L1 single-domain antibody Fc fusion protein (Nb-Fc) was added in a gradient of 184nM, 91.8nM, 45.9nM, 22.9nM, 11.5nM, 5.74nM, 2.87nM, 1.43nM, 0.72nM, 0.36nM, 0.18nM, 0.09nM for each sample, 100uL was added to each sample, 3ug hPD-1(ECD) -Fc-Biotin was added to all samples at the same time, and incubation was carried out at 4 ℃ for 20 min; (2) washing the cells with PBS for 2 times, adding SA-PE from eBioscience, incubating at 4 deg.C for 20min, washing the cells with PBS for 2 times, detecting with flow cytometer (BDFACS Calibur), and using graphpad prismAnd 6, processing data by software.
The results are shown in FIG. 3: bispecific antibodies of different structures have reduced activity of blocking the PD1/PD-L1 interaction compared with the original PD-L1 single-domain antibody, wherein the blocking activity of the double-anti-C is closer to that of a control antibody, and the double-anti-C (IC) is more similar to that of the control antibody506.397nM) has better blocking activity on PD-1/PD-L1 than that of double-antibody D (IC)50＝10.04nM)。
The results show that the activity loss of the PD-1/PD-L1 blocking activity of the double-antibody D is more serious compared with that of the PD-L1 monoclonal antibody, the double-antibody C better retains the blocking activity of the PD-1/PD-L1 and the CD47/SIRPa blocking activity, and the double-antibody C is selected for subsequent research.
Example 4: affinity assay for PD-L1/CD47 and bispecific antibodies
The binding kinetics of the double antibody C to the recombinant human PD-L1 is determined by a biofilm interference technique (Bio-layer interference BLI) and a Fortebio Red96 instrument, and the specific method is as follows:
(1) affinity detection of diabody C with CD 47: test antibodies were diluted in PBST from 100nM with a gradient. The antigenic proteins hCD47(ECD) -Fc and Fc were diluted to 30ug/mL, respectively. Setting the operating conditions of the instrument: the temperature was 30 ℃ and Shake speed 1000 rpm. Capturing the antibody by using a probe coated with ProteinA for 180 s; binding to the antigen at the gradient dilution for 120 s; dissociation time 120 s; 10mM glycine (pH 1.7) was regenerated 3 times for 5s each time. On-machine detection was performed using ForteBio's Octet System.
(2) Affinity detection of diabody C with PD-L1: test antibodies were diluted in PBST from 200nM with a gradient. Antigenic proteins hPD-L1(ECD) -Fc and Fc were diluted to 40ug/mL, respectively. Setting the operating conditions of the instrument: the temperature was 30 ℃ and Shake speed 1000 rpm. Capturing the antibody by using a probe coated with Protein A for 180 s; binding to the antigen at the gradient dilution for 70 s; dissociation time 120 s; 10mM glycine (pH 1.7) was regenerated 3 times for 5s each time. On-machine detection was performed using ForteBio's Octet System.
The detection results are as follows: as shown in FIG. 4A, the affinity of the control antibody CD47 Nb-Fc was 6.13E-09M, and as shown in FIG. 4B, the affinity of the diabody C-CD47 was 6.15E-09M, indicating that the diabody C-CD47 has the same affinity as the control antibody CD47 Nb-Fc. The affinity of the control antibody PD-L1 Nb-Fc was 2.65E-08M as shown in fig. 4C, and the affinity of the diabody C-PD-L1 was 2.66E-08M as shown in fig. 4D, indicating that the diabody C-PD-L1 also had the same affinity as the control antibody PD-L1 Nb-Fc.
Example 5: activity detection of PD-L1/CD47 bispecific antibody for simultaneously binding double targets
And (3) detecting whether the candidate double-antibody C can be simultaneously combined with the double-target antigen protein by using an ELISA method. The specific process is as follows: (1) with NaHCO respectively3CD47 protein or PD-L1 protein is diluted by the solution, the concentration is 1ug/mL, 100uL is coated in an enzyme label plate, and the temperature is kept overnight at 4 ℃; (2) PBST washing for 5 times; (3) blocking 300uL of 1% BSA at 37 ℃ for 2h per well; (4) PBST washing for 5 times; (5) incubating 100ul of diluted double antibody C per well at 37 ℃ for 1 h; (6) PBST washing 5 times, each hole is added with 100uL PD-L1-biotin or CD47-biotin samples and incubated for 1h at 37 ℃; (7) PBST was washed 5 times, added 100ul SA-HRP (1:5000PBS) and incubated at 37 ℃ for 1 h; (8) PBST is washed for 5 times, 100uL of TMB color development liquid is added into each hole, and the reaction is carried out for 5-7min at room temperature in a dark place; (9) plus 50ul 2M H2SO4The reaction was stopped and absorbance was read at 450 nm.
Results as shown in fig. 5A and 5B, fig. 5A is the result of detection when CD47 was coated, and fig. 5B is the result of detection when PD-L1 was coated, indicating that the anti-PD-L1/CD 47 bispecific antibody C of the present invention can bind to CD47 and PD-L1 simultaneously.
Example 6: functional detection of T cell activation by PD-L1/CD47 bispecific antibody
The detection method comprises the following steps: (1) 50uL of human PBMC cells were added to 96-well cell culture plates, 1X10 per well5(ii) individual cells; (2) 100uL of the antibody (final concentration: 100nM, 10nM) and 50uL of SEB (final concentration: 0.1ug/mL) were added to the corresponding wells, respectively; (3)37 ℃ and 5% CO2Culturing for 72 h; (4) and (3) detecting the content of IL-2 in the supernatant by using a BDIL-2ELISA kit.
The results are shown in fig. 6A and 6B, and the content of IL-2 in the experimental group and the control group is significantly higher than that in the negative control group, indicating that in Donor1 and Donor2, both the candidate diabody C and the control antibody PD-L1 Nb-Fc exhibit significant activation effect on T cells.
Example 7: detection of phagocytosis of tumor cells by macrophages induced by PD-L1/CD47 bispecific antibody
The specific process is as follows: (1) PBMCs were induced in RPMI1640 (10% FBS) containing 40ng/ml M-CSF for 9-12 days and macrophages were harvested. (2) Jurkat cells were labeled with CFSE (final concentration: 1.5uM), and macrophage was labeled with eFlour670 (final concentration: 1.25 uM). (3) The macrophage (1E5, 100uL) and Jurkat cells (3E5, 50uL) were mixed well, and 50uL of diluted antibody (final concentration 30ug/mL, 0.3ug/mL) was added. (4)37 ℃ and 5% CO2 for 3 h. (5) The cells in each well were transferred to a 1.5mL EP tube, after which each well was washed with 200uL of PBS, and then the washed PBS was transferred to a corresponding 1.5mL EP tube. (6) Centrifuge at 3000rpm at 4 ℃ for 4min, add 500uL PBS/tube, and resuspend. (7) Centrifuge at 3000rpm at 4 ℃ for 4min, add 200uL PBS/well, resuspend, flow assay.
The results are shown in FIG. 7: both the control antibody CD47 Nb-Fc and the candidate diabody C were effective in promoting macrophage phagocytosis of Jurkat cells.
Example 8: agglutination detection of human erythrocytes by PD-L1/CD47 bispecific antibody
Candidate double anti C, CD47 single domain antibody, positive control antibody B6H12 (sequence source see WO2011143624A2), positive control antibody Hu5F9, and negative control (IgG4) were diluted in 4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 3.906nM, 0.976nM, 0nM, respectively, in gradient. The antibodies diluted in the gradient were added to human erythrocyte suspensions (2%) and reacted overnight at 37 ℃ before observing the results.
The results are shown in fig. 8A and 8B: hu5F9 was able to significantly cause human red blood cell agglutination, B6H12 caused a slight red blood cell agglutination reaction at lower antibody concentrations, while the CD47 single domain antibody and the candidate diabody C did not cause human red blood cell agglutination.
Example 9: stability assay for PD-L1/CD47 bispecific antibodies
The specific method comprises the following steps: 1mg/mL of the double anti-C sample is filtered into a new centrifuge tube using a 0.22um needle filter in a solution of 1xPBS, pH 7.0. The samples were dispensed into EP tubes and placed in the following conditions, each in a respective incubator (see table 2 below). And when the treatment time is up, taking out a sample, and carrying out SEC-HPLC on the sample to detect the purity of the sample.
TABLE 2 stability assay conditions for PD-L1/CD47 bispecific antibodies
Figure BDA0002254258610000201
The SEC detection results are shown in fig. 9A and 9B: the purity of the double-antibody C is not obviously changed after 15 days of treatment, and the double-antibody C can be stably stored at 4 ℃ under the non-preparation condition; and at the accelerated condition of 25 ℃, the double-resistant C is partially degraded, and can be improved subsequently by adjusting the formula of the preparation.
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-L1/CD 47 bispecific antibody and use thereof
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Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
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Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys
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Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
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Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
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Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg
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Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Thr Ser Asp
20 25 30
Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val
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Ala Leu Ile Tyr Thr Pro Gly Ala Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Ser Thr Val Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
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Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys
50 55 60
Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg
100 105 110
Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser
115 120 125
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
130 135 140
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
145 150 155 160
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
165 170 175
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
180 185 190
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
195 200 205
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
210 215 220
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
225 230 235 240
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
245 250 255
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
260 265 270
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
275 280 285
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
290 295 300
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
305 310 315 320
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
325 330 335
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
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Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
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Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly
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Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
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Gly Lys Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly Ala Ser
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Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp
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Asn Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
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Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys Ser Leu
465 470 475 480
Arg Leu Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Thr Ser Asp
20 25 30
Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val
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Ala Leu Ile Tyr Thr Pro Gly Ala Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Ser Thr Val Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
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Ala Ala Arg Arg Gly Ala Cys Ser Leu Arg Leu Pro Phe Phe Tyr Trp
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Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro
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Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
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Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
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Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
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Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
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Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gln
340 345 350
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
355 360 365
Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser Cys
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Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala
385 390 395 400
Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Ala
405 410 415
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
420 425 430
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
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Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg Asp
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Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser
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Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys
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Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
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Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg
100 105 110
Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
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Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser
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Pro Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu
180 185 190
Gly Val Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser
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Val Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
210 215 220
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ala Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser
245 250 255
Ala Arg Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
260 265 270
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
275 280 285
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
290 295 300
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
305 310 315 320
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
325 330 335
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
340 345 350
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
355 360 365
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
370 375 380
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
385 390 395 400
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
405 410 415
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
420 425 430
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
435 440 445
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
450 455 460
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
465 470 475 480
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
485 490 495
Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
500 505 510
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser
515 520 525
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
530 535 540
Ala Ser Gly Tyr Ala Tyr Thr Ser Asp Cys Met Gly Trp Phe Arg Gln
545 550 555 560
Thr Pro Gly Lys Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly
565 570 575
Ala Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
580 585 590
Gln Asp Asn Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg
595 600 605
Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys
610 615 620
Ser Leu Arg Leu Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr
625 630 635 640
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
645 650 655
Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly
660 665 670
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
675 680 685
Gly Tyr Ala Tyr Thr Ser Asp Cys Met Gly Trp Phe Arg Gln Thr Pro
690 695 700
Gly Lys Gly Leu Glu Gly Val Ala Leu Ile Tyr Thr Pro Gly Ala Ser
705 710 715 720
Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp
725 730 735
Asn Ser Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
740 745 750
Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg Gly Ala Cys Ser Leu
755 760 765
Arg Leu Pro Phe Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
770 775 780
Ser
785
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Thr Ser Asp
20 25 30
Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Leu Ile Tyr Thr Pro Gly Ala Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Arg Arg Gly Ala Cys Ser Leu Arg Leu Pro Phe Phe Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
130 135 140
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
145 150 155 160
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Thr Ser Asp Cys Met
165 170 175
Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Gly Val Ala Leu
180 185 190
Ile Tyr Thr Pro Gly Ala Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly
195 200 205
Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Ser Thr Val Tyr Leu Gln
210 215 220
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
225 230 235 240
Arg Arg Gly Ala Cys Ser Leu Arg Leu Pro Phe Phe Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
260 265 270
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
275 280 285
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
290 295 300
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
305 310 315 320
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
325 330 335
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
340 345 350
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
355 360 365
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
370 375 380
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
385 390 395 400
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
405 410 415
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
420 425 430
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
435 440 445
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
450 455 460
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
465 470 475 480
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gln Val Gln
485 490 495
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
500 505 510
Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser Cys Met Gly
515 520 525
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Phe Thr
530 535 540
Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Ala Arg Phe
545 550 555 560
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
565 570 575
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Phe
580 585 590
Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg Asp Lys Tyr
595 600 605
Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
610 615 620
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
625 630 635 640
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
645 650 655
Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser Cys
660 665 670
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala
675 680 685
Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Ala
690 695 700
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
705 710 715 720
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
725 730 735
Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg Asp
740 745 750
Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
755 760 765
<210> 8
<211> 378
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcaccg cctccggcta caacctgtcc ccctcctgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccttc accgacgccg acggctccac caggtacgcc 180
gactccgtga aggccaggtt caccatctcc agggacaact ccaagaacac cctgtacctg 240
cagatgaact ccctgagggc cgaggacacc gccgtgtact actgcgccgc cgacttcttc 300
tcctactgct ccgtggtgtt cagggcctcc gccagggaca agtacagggg ccagggcacc 360
ctggtgaccg tgtcctcc 378
<210> 9
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggcggcggcg gctccggcgg cggcggctcc ggcggcggcg gctccggcgg cggcggctcc 60
<210> 10
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctacacc tccgactgca tgggctggtt caggcagacc 120
cccggcaagg gcctggaggg cgtggccctg atctacaccc ccggcgcctc caccaactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca actccaagtc caccgtgtac 240
ctgcagatga actccctgag ggccgaggac accgccatgt actactgcgc cgccaggagg 300
ggcgcctgct ccctgaggct gcccttcttc tactggggcc agggcaccct ggtgaccgtg 360
tcctcc 366
<210> 11
<211> 1491
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcaccg cctccggcta caacctgtcc ccctcctgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccttc accgacgccg acggctccac caggtacgcc 180
gactccgtga aggccaggtt caccatctcc agggacaact ccaagaacac cctgtacctg 240
cagatgaact ccctgagggc cgaggacacc gccgtgtact actgcgccgc cgacttcttc 300
tcctactgct ccgtggtgtt cagggcctcc gccagggaca agtacagggg ccagggcacc 360
ctggtgaccg tgtcctccga gtccaagtac ggccccccct gccccccctg ccccgccccc 420
gagttcctgg gcggcccctc cgtgttcctg ttccccccca agcccaagga caccctgatg 480
atctccagga cccccgaggt gacctgcgtg gtggtggacg tgtcccagga ggaccccgag 540
gtgcagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccagg 600
gaggagcagt tcaactccac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac 660
tggctgaacg gcaaggagta caagtgcaag gtgtccaaca agggcctgcc ctcctccatc 720
gagaagacca tctccaaggc caagggccag cccagggagc cccaggtgta caccctgccc 780
ccctcccagg aggagatgac caagaaccag gtgtccctga cctgcctggt gaagggcttc 840
tacccctccg acatcgccgt ggagtgggag tccaacggcc agcccgagaa caactacaag 900
accacccccc ccgtgctgga ctccgacggc tccttcttcc tgtactccag gctgaccgtg 960
gacaagtcca ggtggcagga gggcaacgtg ttctcctgct ccgtgatgca cgaggccctg 1020
cacaaccact acacccagaa gtccctgtcc ctgtccctgg gcaagggcgg cggcggctcc 1080
ggcggcggcg gctccggcgg cggcggctcc ggcggcggcg gctcccaggt gcagctgcag 1140
gagtccggcg gcggcctggt gcagcccggc ggctccctga ggctgtcctg cgccgcctcc 1200
ggctacgcct acacctccga ctgcatgggc tggttcaggc agacccccgg caagggcctg 1260
gagggcgtgg ccctgatcta cacccccggc gcctccacca actacgccga ctccgtgaag 1320
ggcaggttca ccatctccca ggacaactcc aagtccaccg tgtacctgca gatgaactcc 1380
ctgagggccg aggacaccgc catgtactac tgcgccgcca ggaggggcgc ctgctccctg 1440
aggctgccct tcttctactg gggccagggc accctggtga ccgtgtcctc c 1491
<210> 12
<211> 1491
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctacacc tccgactgca tgggctggtt caggcagacc 120
cccggcaagg gcctggaggg cgtggccctg atctacaccc ccggcgcctc caccaactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca actccaagtc caccgtgtac 240
ctgcagatga actccctgag ggccgaggac accgccatgt actactgcgc cgccaggagg 300
ggcgcctgct ccctgaggct gcccttcttc tactggggcc agggcaccct ggtgaccgtg 360
tcctccgagt ccaagtacgg ccccccctgc cccccctgcc ccgcccccga gttcctgggc 420
ggcccctccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat ctccaggacc 480
cccgaggtga cctgcgtggt ggtggacgtg tcccaggagg accccgaggt gcagttcaac 540
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccaggga ggagcagttc 600
aactccacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 660
aaggagtaca agtgcaaggt gtccaacaag ggcctgccct cctccatcga gaagaccatc 720
tccaaggcca agggccagcc cagggagccc caggtgtaca ccctgccccc ctcccaggag 780
gagatgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta cccctccgac 840
atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 900
gtgctggact ccgacggctc cttcttcctg tactccaggc tgaccgtgga caagtccagg 960
tggcaggagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1020
acccagaagt ccctgtccct gtccctgggc aagggcggcg gcggctccgg cggcggcggc 1080
tccggcggcg gcggctccgg cggcggcggc tcccaggtgc agctgcagga gtccggcggc 1140
ggcctggtgc agcccggcgg ctccctgagg ctgtcctgca ccgcctccgg ctacaacctg 1200
tccccctcct gcatgggctg gttcaggcag gcccccggca agggcctgga gggcgtggcc 1260
ttcaccgacg ccgacggctc caccaggtac gccgactccg tgaaggccag gttcaccatc 1320
tccagggaca actccaagaa caccctgtac ctgcagatga actccctgag ggccgaggac 1380
accgccgtgt actactgcgc cgccgacttc ttctcctact gctccgtggt gttcagggcc 1440
tccgccaggg acaagtacag gggccagggc accctggtga ccgtgtcctc c 1491
<210> 13
<211> 2355
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcaccg cctccggcta caacctgtcc ccctcctgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccttc accgacgccg acggctccac caggtacgcc 180
gactccgtga aggccaggtt caccatctcc agggacaact ccaagaacac cctgtacctg 240
cagatgaact ccctgagggc cgaggacacc gccgtgtact actgcgccgc cgacttcttc 300
tcctactgct ccgtggtgtt cagggcctcc gccagggaca agtacagggg ccagggcacc 360
ctggtgaccg tgtcctccgg cggcggcggc tccggcggcg gcggctccgg cggcggcggc 420
tccggcggcg gcggctccca ggtgcagctg caggagtccg gcggcggcct ggtgcagccc 480
ggcggctccc tgaggctgtc ctgcaccgcc tccggctaca acctgtcccc ctcctgcatg 540
ggctggttca ggcaggcccc cggcaagggc ctggagggcg tggccttcac cgacgccgac 600
ggctccacca ggtacgccga ctccgtgaag gccaggttca ccatctccag ggacaactcc 660
aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggacaccgc cgtgtactac 720
tgcgccgccg acttcttctc ctactgctcc gtggtgttca gggcctccgc cagggacaag 780
tacaggggcc agggcaccct ggtgaccgtg tcctccgagt ccaagtacgg ccccccctgc 840
cccccctgcc ccgcccccga gttcctgggc ggcccctccg tgttcctgtt cccccccaag 900
cccaaggaca ccctgatgat ctccaggacc cccgaggtga cctgcgtggt ggtggacgtg 960
tcccaggagg accccgaggt gcagttcaac tggtacgtgg acggcgtgga ggtgcacaac 1020
gccaagacca agcccaggga ggagcagttc aactccacct acagggtggt gtccgtgctg 1080
accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gtccaacaag 1140
ggcctgccct cctccatcga gaagaccatc tccaaggcca agggccagcc cagggagccc 1200
caggtgtaca ccctgccccc ctcccaggag gagatgacca agaaccaggt gtccctgacc 1260
tgcctggtga agggcttcta cccctccgac atcgccgtgg agtgggagtc caacggccag 1320
cccgagaaca actacaagac cacccccccc gtgctggact ccgacggctc cttcttcctg 1380
tactccaggc tgaccgtgga caagtccagg tggcaggagg gcaacgtgtt ctcctgctcc 1440
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgtccct gtccctgggc 1500
aagggcggcg gcggctccgg cggcggcggc tccggcggcg gcggctccgg cggcggcggc 1560
tcccaggtgc agctgcagga gtccggcggc ggcctggtgc agcccggcgg ctccctgagg 1620
ctgtcctgcg ccgcctccgg ctacgcctac acctccgact gcatgggctg gttcaggcag 1680
acccccggca agggcctgga gggcgtggcc ctgatctaca cccccggcgc ctccaccaac 1740
tacgccgact ccgtgaaggg caggttcacc atctcccagg acaactccaa gtccaccgtg 1800
tacctgcaga tgaactccct gagggccgag gacaccgcca tgtactactg cgccgccagg 1860
aggggcgcct gctccctgag gctgcccttc ttctactggg gccagggcac cctggtgacc 1920
gtgtcctccg gcggcggcgg ctccggcggc ggcggctccg gcggcggcgg ctccggcggc 1980
ggcggctccc aggtgcagct gcaggagtcc ggcggcggcc tggtgcagcc cggcggctcc 2040
ctgaggctgt cctgcgccgc ctccggctac gcctacacct ccgactgcat gggctggttc 2100
aggcagaccc ccggcaaggg cctggagggc gtggccctga tctacacccc cggcgcctcc 2160
accaactacg ccgactccgt gaagggcagg ttcaccatct cccaggacaa ctccaagtcc 2220
accgtgtacc tgcagatgaa ctccctgagg gccgaggaca ccgccatgta ctactgcgcc 2280
gccaggaggg gcgcctgctc cctgaggctg cccttcttct actggggcca gggcaccctg 2340
gtgaccgtgt cctcc 2355
<210> 14
<211> 2355
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctacacc tccgactgca tgggctggtt caggcagacc 120
cccggcaagg gcctggaggg cgtggccctg atctacaccc ccggcgcctc caccaactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca actccaagtc caccgtgtac 240
ctgcagatga actccctgag ggccgaggac accgccatgt actactgcgc cgccaggagg 300
ggcgcctgct ccctgaggct gcccttcttc tactggggcc agggcaccct ggtgaccgtg 360
tcctccggcg gcggcggctc cggcggcggc ggctccggcg gcggcggctc cggcggcggc 420
ggctcccagg tgcagctgca ggagtccggc ggcggcctgg tgcagcccgg cggctccctg 480
aggctgtcct gcgccgcctc cggctacgcc tacacctccg actgcatggg ctggttcagg 540
cagacccccg gcaagggcct ggagggcgtg gccctgatct acacccccgg cgcctccacc 600
aactacgccg actccgtgaa gggcaggttc accatctccc aggacaactc caagtccacc 660
gtgtacctgc agatgaactc cctgagggcc gaggacaccg ccatgtacta ctgcgccgcc 720
aggaggggcg cctgctccct gaggctgccc ttcttctact ggggccaggg caccctggtg 780
accgtgtcct ccgagtccaa gtacggcccc ccctgccccc cctgccccgc ccccgagttc 840
ctgggcggcc cctccgtgtt cctgttcccc cccaagccca aggacaccct gatgatctcc 900
aggacccccg aggtgacctg cgtggtggtg gacgtgtccc aggaggaccc cgaggtgcag 960
ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 1020
cagttcaact ccacctacag ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg 1080
aacggcaagg agtacaagtg caaggtgtcc aacaagggcc tgccctcctc catcgagaag 1140
accatctcca aggccaaggg ccagcccagg gagccccagg tgtacaccct gcccccctcc 1200
caggaggaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1260
tccgacatcg ccgtggagtg ggagtccaac ggccagcccg agaacaacta caagaccacc 1320
ccccccgtgc tggactccga cggctccttc ttcctgtact ccaggctgac cgtggacaag 1380
tccaggtggc aggagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1440
cactacaccc agaagtccct gtccctgtcc ctgggcaagg gcggcggcgg ctccggcggc 1500
ggcggctccg gcggcggcgg ctccggcggc ggcggctccc aggtgcagct gcaggagtcc 1560
ggcggcggcc tggtgcagcc cggcggctcc ctgaggctgt cctgcaccgc ctccggctac 1620
aacctgtccc cctcctgcat gggctggttc aggcaggccc ccggcaaggg cctggagggc 1680
gtggccttca ccgacgccga cggctccacc aggtacgccg actccgtgaa ggccaggttc 1740
accatctcca gggacaactc caagaacacc ctgtacctgc agatgaactc cctgagggcc 1800
gaggacaccg ccgtgtacta ctgcgccgcc gacttcttct cctactgctc cgtggtgttc 1860
agggcctccg ccagggacaa gtacaggggc cagggcaccc tggtgaccgt gtcctccggc 1920
ggcggcggct ccggcggcgg cggctccggc ggcggcggct ccggcggcgg cggctcccag 1980
gtgcagctgc aggagtccgg cggcggcctg gtgcagcccg gcggctccct gaggctgtcc 2040
tgcaccgcct ccggctacaa cctgtccccc tcctgcatgg gctggttcag gcaggccccc 2100
ggcaagggcc tggagggcgt ggccttcacc gacgccgacg gctccaccag gtacgccgac 2160
tccgtgaagg ccaggttcac catctccagg gacaactcca agaacaccct gtacctgcag 2220
atgaactccc tgagggccga ggacaccgcc gtgtactact gcgccgccga cttcttctcc 2280
tactgctccg tggtgttcag ggcctccgcc agggacaagt acaggggcca gggcaccctg 2340
gtgaccgtgt cctcc 2355

Claims (10)

1. A bispecific antibody, wherein said bispecific antibody comprises: anti-PD-L1 single domain antibodies and anti-CD 47 single domain antibodies.
2. The bispecific antibody of claim 1, wherein said bispecific antibody comprises 2 anti-PD-L1 single domain antibodies and anti-CD 47 single domain antibodies.
3. The bispecific antibody of claim 1, wherein the bispecific antibody has a structure from N-terminus to C-terminus represented by formula i (a) or i (b):
P-L1-P-L2-Fc-L3-B-L4-B formula I (a)
B-L1-B-L2-Fc-L3-P-L4-P formula I (B)
Wherein the content of the first and second substances,
"-" is a peptide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is an anti-PD-L1 single domain antibody,
b is an anti-CD 47 single domain antibody, and
fc is the Fc segment of the antibody.
4. A bispecific fusion protein characterized in that said bispecific fusion protein is a dimer formed by two bispecific antibodies of claim 1.
5. A polynucleotide encoding the bispecific antibody of claim 1 or the bispecific fusion protein of claim 4.
6. An expression vector comprising the polynucleotide of claim 5.
7. A host cell comprising the expression vector of claim 6, or having the polynucleotide of claim 5 integrated into its genome;
alternatively, the host cell expresses the bispecific antibody of claim 1.
8. A method of making a bispecific antibody comprising the steps of:
(a) culturing the host cell of claim 7 under suitable conditions, thereby obtaining a culture comprising the bispecific antibody; and
(b) purifying and/or isolating the culture obtained in step (a) to obtain said bispecific antibody.
9. An immunoconjugate, comprising:
(a) the bispecific antibody of claim 1 or the bispecific fusion protein of claim 4; 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, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein or VLP, or a combination thereof.
10. Use of the bispecific antibody of claim 1 or the bispecific fusion protein of claim 4 for the preparation of (a) a reagent, kit for the detection of a PD-L1 and/or CD47 molecule; (b) a Chinese medicinal composition for preventing and treating tumor.
CN201911046449.0A 2019-10-30 2019-10-30 anti-PD-L1/CD 47 bispecific antibody and application thereof Active CN112745392B (en)

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WO2022235127A1 (en) * 2021-05-07 2022-11-10 주식회사 이뮨온시아 Bispecific antibody specifically binding to cd47 and pd-l1
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WO2023056970A1 (en) * 2021-10-09 2023-04-13 Hutchmed Limited Bispecific antibodies specifically binding to cd47 and her2, and uses thereof
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WO2023224412A1 (en) * 2022-05-19 2023-11-23 (주)샤페론 Bispecific humanized single domain antibody to pd-l1 and cd47, and use thereof

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