CN112442122B - Blocking type PD-1 nano antibody and its coding sequence and use - Google Patents

Blocking type PD-1 nano antibody and its coding sequence and use Download PDF

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CN112442122B
CN112442122B CN202010915175.0A CN202010915175A CN112442122B CN 112442122 B CN112442122 B CN 112442122B CN 202010915175 A CN202010915175 A CN 202010915175A CN 112442122 B CN112442122 B CN 112442122B
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万亚坤
朱敏
盖军伟
李光辉
沈晓宁
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Shanghai Novamab Biopharmaceuticals Co Ltd
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Abstract

The invention provides a blocking type PD-1 nano antibody and a coding sequence and application thereof. Specifically, the invention provides a specific nano antibody of anti-human PD-1 and a VHH chain thereof. The invention also provides a coding sequence for coding the nano antibody or the VHH chain thereof, a corresponding expression vector, a host cell capable of expressing the nano antibody and a production method of the nano antibody. The nano antibody has good biological activity of blocking a PD-1/PD-L1 signal channel, and effectively resists the growth of tumors. Can be used for detection and targeted therapy of PD-1.

Description

Blocking type PD-1 nano antibody and its coding sequence and use
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, in particular to a blocking type PD-1 nano antibody and a coding sequence and application thereof.
Background
Programmed cell death protein 1 (PD-1) is a cell surface receptor, also known as CD279, a member of the CD28 family, and functions in the immune system to regulate T cells. The human PD-1 gene is located at chromosome 2q 37. The full-length PD-1 is an I-type transmembrane protein containing 288 amino acids, mainly comprises an extracellular immunoglobulin variable region (IgV) like structure domain (amino acids 21-170), a hydrophobic transmembrane region (amino acids 171 and 191) and an intracellular region (amino acids 192 and 288), and has 60 percent of homology with the murine PD-1. During thymic development, PD-1 is expressed on the surface of thymocytes that are double negative to CD4 and CD8, and is predominantly expressed on the surface of activated T cells, B cells, NKT cells, and monocytes.
PD-1 is considered to be a co-inhibitory receptor. The binding ligand comprises: programmed cell death ligand 1(programmed cell death 1ligand 1, PD-L1, B7-H1) and programmed cell death ligand 2(programmed cell death 1ligand 2, PD-L2, B7-DC). By combining PD-L1 or PD-L2 with PD-1, PD-1 can down-regulate the activation level of T cells, inhibit the proliferation of the T cells and simultaneously down-regulate the expression of an anti-apoptotic molecule Bcl-xL and cytokines in immune cells and the function of an mTOR (mammalian target of rapamycin) pathway in the immune cells. Thus, PD-1, which is induced to be expressed by T cells upon activation in peripheral tissues and tumor microenvironments, may respond to chronic inflammation or tumor cell surface antigen expression, bind to tumor cell or antigen presenting cell surface PD-L1, and function as an immune checkpoint for suppressing a sustained immune response and preventing potential damage to normal tissues.
T cell responses to tumor surface antigens may be deregulated by tumor cells, and when tumor cells attempt to evade immunodetection by hijacking and disrupting the homeostatic immune checkpoint signaling pathway, T cell responses to these antigens may be deregulated. For example, binding of PD-L1 to PD-1 down-regulates anti-tumor T cell activity and promotes immune evasion. In human melanoma, tumor infiltrating T lymphocytes (TILs) highly upregulate PD-1 and exhibit impaired effector cytokine production compared to PD-1-TILs and peripheral blood lymphocytes, which may indicate a T cell exhaustion state. In addition, T cell activation produces a variety of cytokines, such as IL-2, TNF-a, and IFN- γ, among others. IFN-gamma production continued to induce expression up-regulation of PD-1. Resulting in self-inhibition and thus in the induction of immune resistance. Therefore, successful blocking of the binding of PD-1 to its ligand can be considered as an effective approach for tumor therapy.
PD-1/PD-L1 immunotherapy kills cancer cells by blocking the PD-1/PD-L1 signaling pathway, has the potential to treat multiple types of tumors, and substantially improves the overall survival of patients. By the end of 2018, the FDA approved 6 PD-1/L1 monoclonal antibody drugs, 3 PD-1 inhibitors: keytruda by Sanofi, by Keytruda by Sansto, Opdivo by BMS, by Regeneron. In addition, the domestic PD-1 drugs: terepril monoclonal antibody (Tuiyi) of Junchengshi organism, Xindilizumab (Darbu) of Xindaji organism, and Carrilizumab (Airitol) of Henrie medicine are approved for sale.
As a representative of new antibody drugs, the nano antibody is being widely applied to research and development of new drugs, and the nano antibody drug has many advantages and will play a more important role in immunotherapy of tumors. However, satisfactory nanobodies against PD-1 are lacking in the art. Therefore, the development of a new effective nano antibody against PD-1, blocking the mutual combination of PD-1 and PD-L1, and thus applying to the immunotherapy of tumors is urgently needed in the field.
Disclosure of Invention
The invention aims to provide a blocking type PD-1 nano antibody and a coding sequence and application thereof.
Specifically, the invention aims to provide a nano antibody which is effective against PD-1, can effectively block the interaction of PD-1/PD-L1 and has good anti-tumor activity.
In a first aspect of the present invention, there is provided a VHH chain of an anti-PD-1 nanobody, the VHH chain comprising a framework region FR and complementarity determining region CDRs comprising CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3.
In another preferred embodiment, the framework region FR comprises:
(a) FR1 shown by SEQ ID NO. 4, FR2 shown by SEQ ID NO. 5, FR3 shown by SEQ ID NO. 6, and FR4 shown by SEQ ID NO. 7; or
(b) FR1 shown in SEQ ID NO. 10, FR2 shown in SEQ ID NO. 11, FR3 shown in SEQ ID NO. 12, and FR4 shown in SEQ ID NO. 13.
In another preferred embodiment, the VHH chain of the anti-PD-1 nano antibody is shown as SEQ ID NO. 8 or 14.
In another preferred example, the PD-1 is human PD-1.
In addition, the heavy chain variable region of the anti-human PD-1 antibody is provided, and comprises a CDR1 shown in SEQ ID NO. 1, a CDR2 shown in SEQ ID NO. 2 and a CDR3 shown in SEQ ID NO. 3.
In a second aspect of the present invention, there is provided an anti-PD-1 nanobody, which is a nanobody against a PD-1 epitope and has a VHH chain having an amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 14.
In another preferred embodiment, the IC of the anti-PD-1 nanobody50Less than 2ug/mL, preferably less than 1.5 ug/mL.
In a third aspect of the invention, there is provided an anti-PD-1 multivalent antibody comprising 2, 3 or 4 VHH chains of an anti-PD-1 nanobody according to the first aspect of the invention or an anti-PD-1 nanobody according to the second aspect of the invention.
In another preferred embodiment, the multivalent antibody has a structure represented by formula II or formula III from N-terminus to C-terminus:
P-L' -P-Fc formula II
P-L '-P-L' -P-Fc formula II
Wherein,
"-" is a peptide bond;
l' is a component of the joint,
p is an anti-PD-1 nano antibody,
fc is the Fc segment of the antibody.
In another preferred example, the sequence of L' is (4GS) n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), and preferably n is 2 or 4.
In another preferred embodiment, the sequence of Fc is shown in the 281-509 th position of SEQ ID NO. 20.
In another preferred embodiment, the amino acid sequence of the multivalent antibody is shown in SEQ ID NO. 18-20, 24-26.
In another preferred embodiment, the amino acid sequence of the multivalent antibody is as shown in SEQ ID No. 20 or 26.
In another preferred embodiment, the multivalent antibody may form a dimer.
In another preferred embodiment, the dimer is a tetravalent antibody or a hexavalent antibody.
In a fourth aspect of the invention, there is provided a bispecific antibody comprising: an anti-PD-1 nanobody and a secondary antibody,
wherein the CDR of the anti-PD-1 nano antibody comprises:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3.
In another preferred embodiment, the second antibody comprises a nanobody, a single-chain antibody, or a double-chain antibody.
In another preferred embodiment, the second antibody is selected from the group consisting of:
4-1BB antibody, CD47 antibody, VEGF antibody, HER2 antibody, EGFR antibody, HER3 antibody, B7H3 antibody, TIGIT antibody, OX-40 antibody, CD40 antibody, PD-L1 antibody, or a combination thereof.
In another preferred embodiment, the bispecific antibody comprises 2 to 4 anti-PD-1 nanobodies, preferably 2 anti-PD-1 nanobodies, and more preferably the 2 anti-PD-1 nanobodies form an anti-PD-1 nanobody dimer.
In another preferred embodiment, the second antibody is a nanobody, and the bispecific antibody comprises 2 to 4 second antibodies, preferably 2 second antibodies, and more preferably 2 second antibodies form a dimer.
In another preferred embodiment, the second antibody is a single chain antibody and the bispecific antibody comprises 1-2 second antibodies.
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 from N-terminus to C-terminus:
P-L1-P-L2-Fc-L3-B-L4-B formula I
Wherein,
"-" is a peptide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is an anti-PD-1 nano antibody,
b is a secondary antibody (nanobody), and
fc is the Fc segment of the antibody.
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, said L2 is a peptide bond.
In a fifth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the VHH chain of an anti-PD-1 nanobody according to the first aspect of the invention, or an anti-PD-1 nanobody according to the second aspect of the invention, an anti-PD-1 multivalent antibody according to the third aspect of the invention, or a bispecific antibody according to the fourth aspect of the invention.
In another preferred embodiment, the polynucleotide has the nucleotide sequence shown in SEQ ID NO.9 or 15.
In another preferred embodiment, the polynucleotide comprises DNA or RNA.
In a sixth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the fifth 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 seventh aspect of the invention, there is provided a host cell comprising an expression vector according to the sixth aspect of the invention, or having a polynucleotide according to the fifth aspect of the invention integrated into its genome.
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 an eighth aspect of the present invention, there is provided a method of producing an anti-PD-1 nanobody or an anti-PD-1 multivalent antibody, comprising the steps of:
(a) culturing a host cell according to the seventh aspect of the invention under suitable conditions, thereby obtaining a culture comprising the anti-PD-1 nanobody; and
(b) (ii) purifying and/or isolating the culture obtained in step (i) to obtain said anti-PD-1 nanobody.
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 ninth aspect of the invention, there is provided an immunoconjugate comprising:
(a) a VHH chain of an anti-PD-1 nanobody of the first aspect, an anti-PD-1 nanobody of the second aspect, an anti-PD-1 multivalent antibody of the third aspect of the invention, or a bispecific antibody of the fourth 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, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or nanoparticles in any form.
In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g. bivalent) VHH chain of an anti-PD-1 nanobody according to the first aspect of the invention, an anti-PD-1 nanobody according to the second aspect of the invention, an anti-PD-1 multivalent antibody according to the third aspect of the invention.
In another preferred embodiment, the multivalent refers to a VHH chain of an anti-PD-1 nanobody according to the first aspect of the invention, an anti-PD-1 nanobody according to the second aspect of the invention, or an anti-PD-1 multivalent antibody according to the third aspect of the invention, comprising multiple repeats in the amino acid sequence of the immunoconjugate.
In a tenth aspect of the invention, there is provided the use of an anti-PD-1 nanobody according to the second aspect of the invention or an anti-PD-1 multivalent antibody according to the third aspect of the invention, for the preparation of (a) a reagent for the detection of a PD-1 molecule; (b) can be used for preparing medicine for 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 embodiment, the agent for detecting PD-1 molecules is a contrast agent for detecting PD-1 molecules (in vivo).
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 for blocking the interaction of PD-1 and PD-L1.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) a VHH chain according to the first aspect of the invention, an anti-PD-1 nanobody according to the second aspect of the invention, an anti-PD-1 multivalent antibody according to the third aspect of the invention, a bispecific antibody according to the fourth aspect of the invention, or an immunoconjugate according to the ninth 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.
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 in the form of injection.
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 used for preparing a medicament for treating tumors selected from the group consisting of: colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestinal cancer, bone cancer, prostate cancer, cervical cancer, lymph cancer, adrenal tumor, or bladder tumor.
In a twelfth aspect of the invention, there is provided a use of one or more of the anti-PD-1 nanobody of the second aspect of the invention or the anti-PD-1 multivalent antibody of the third aspect of the invention: (i) for detecting human PD-1 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.
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 a thirteenth aspect of the present invention, there is also provided an antibody having: a heavy chain variable region VHH according to the first aspect of the invention.
In another preferred embodiment, the antibody is an antibody specific to PD-1 protein.
In another preferred embodiment, the antibody is a nanobody.
In a fourteenth aspect of the present invention, there is provided a recombinant protein having: (i) a sequence of a VHH according to the first aspect of the invention, a sequence of a nanobody according to the second aspect of the invention, or a sequence of an anti-PD-1 multivalent antibody according to the third 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 a PD-1 protein.
In a fifteenth aspect of the invention there is provided the use of a VHH chain according to the first aspect of the invention, a nanobody according to the second aspect of the invention, an anti-PD-1 multivalent antibody according to the third aspect of the invention, or an immunoconjugate according to the ninth 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 the PD-1 protein in the sample; wherein the medicament is used for treating or preventing tumors expressing PD-L1 protein (i.e. PD-L1 positive).
In another preferred embodiment, the tumor comprises: colorectal cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, lymphoma, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, or adrenal tumor.
In a sixteenth aspect of the present invention, there is provided a method of detecting a PD-1 protein in a sample, the method comprising the steps of: (1) contacting the sample with a nanobody according to the second aspect of the invention or an anti-PD-1 multivalent antibody according to the third aspect of the invention; (2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of the PD-1 protein in the sample.
In a seventeenth aspect of the invention, there is provided a method of treating a disease, the method comprising administering to a subject in need thereof a nanobody according to the second aspect of the invention, an anti-PD-1 multivalent antibody according to the third aspect of the invention, a bispecific antibody according to the fourth aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention.
In another preferred embodiment, the subject comprises a mammal, such as a human.
In the eighteenth aspect of the invention, a PD-1 protein detection reagent is provided, wherein the detection reagent comprises the immunoconjugate of the ninth 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 nineteenth aspect of the present invention, there is provided a kit for detecting a PD-1 protein, the kit comprising the immunoconjugate of the ninth aspect of the present invention or the detection reagent of the eighteenth aspect of the present invention, and instructions.
In another preferred embodiment, the instructions describe that the kit is used for non-invasively detecting PD-1 expression in a test subject.
In a twentieth aspect of the invention, there is provided a use of the immunoconjugate according to the ninth aspect of the invention for the preparation of a contrast agent for the in vivo detection of PD-1 protein.
In another preferred embodiment, the detection is used for the diagnosis or prognosis of cancer.
In a twenty-first aspect of the invention there is provided a CAR-T cell expressing a chimeric antigen receptor CAR, the antigen binding domain of the CAR having an anti-PD-1 nanobody as described in the second aspect of the invention.
In a twenty-second aspect of the invention there is provided a formulation comprising a CAR-T cell according to the twenty-first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, the formulation comprises an injection.
In another preferred embodiment, the CAR-T cells are present in the formulation at a concentration of 1X 103-1×108Individual cells/ml, preferably 1X 104-1×107Individual cells/ml.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the result of SDS-PAGE by gel electrophoresis of PD-1 extracellular domain fusion Fc protein. The results show that: the purity of the obtained PD-1(ECD) -Fc protein reaches more than 90 percent.
FIG. 2 shows the results of library capacity detection of the constructed PD-1 immune library. The library was diluted in gradient and plated to a capacity of 2.2X 109CFU。
FIG. 3 shows the enrichment process of panning nanobodies from PD-1 immune library. The results show that: through six rounds of phage display screening, the PD-1 nanometer antibody phage is enriched by 21.3 times.
FIG. 4 shows the result of construction of PD-1 overexpressing CHOZn cells. The flow cytometry detection result shows that: the constructed CHOZn stable cell strain can highly express human PD-1 and can be used for subsequent research.
FIG. 5 shows the results of flow cytometry for the detection of blocking PD-1 nanobody. The results show that: the nano antibody obtained by screening can block the interaction of PD-1/PD-L1.
FIG. 6 shows IC detection of PD-1 nanobodies by flow cytometry50And (6) obtaining the result. The results show that: i of PD-1 NanobodyC50IC of 1.305ug/mL, control antibody Keytruda50It was 1.339 ug/mL.
FIG. 7 shows EC in flow cytometry for detection of PD-1 nanobodies50And (6) obtaining the result. The results show that: EC of PD-1 Nanobody501.064ug/mL, E of the control antibody KeytrudaC50It was 1.113 ug/mL.
FIG. 8 shows the IC of flow cytometry for detection of humanized PD-1 nanobody50And (6) obtaining the result. The results show that: IC of humanized PD-1 nanobody501.035ug/mL, IC of the control antibody Keytruda50It was 1.169 ug/mL.
FIG. 9 shows the results of the measurement of the biological activity of the humanized antibody using Luciferase reporter assay. The results show that: the humanized PD-1 nano antibody has good biological activity and EC50It was 0.6986 ug/mL.
FIG. 10 shows the results of the bioactivity of humanized multivalent PD-1 nanobody detected using Luciferase reporter assay. The results show that: the blocking activity of the multivalent PD-1 nano antibody is obviously improved compared with that of a bivalent PD-1 nano antibody.
Figure 11 shows the statistical results of tumor volumes for the study of the efficacy of humanized antibodies in a humanized mouse colon cancer model. The results show that: the humanized nanobody (antibody C) had a 91% tumor suppression rate, which is superior to the anti-tumor activity of the control antibody Keytruda.
Figure 12 shows tumor weight statistics for the pharmacodynamic endpoint of the humanized antibody in the humanized mouse colon cancer model. The results show that: the humanized nanobody (antibody C) and the Keytruda control antibody group have significantly lower tumor weights than the negative control group, and the mice with the (antibody C) administered have smaller tumors and more significant efficacy.
Detailed Description
The inventor of the invention has extensively and deeply studied and unexpectedly found a class of anti-PD-1 nano antibody for the first time, and the experimental result shows that the nano antibody of the invention has better binding activity with PD-1 molecules, can block the interaction of PD-1 and PD-L1, and has good anti-tumor activity. The present invention has been completed based on this finding.
Specifically, the invention utilizes the humanized PD-1 antigen protein to immunize camels to obtain a high-quality immune nano antibody gene library. Then coupling the PD-1 protein molecule on an enzyme label plate to display the correct space structure of the PD-1 protein, and screening an immune nano antibody gene library (a camel heavy chain antibody phage display gene library) by using the antigen in the form through a phage display technology so as to obtain the PD-1 specific nano antibody gene. Then the gene is transferred into mammalian cells, thereby obtaining the nano antibody strain which can be efficiently expressed in the mammalian cells. Then, the PD-1 nano antibody with blocking activity is identified by methods such as ELISA, flow cytometry, luciferase reporter gene detection system and the like, and the candidate antibody has obvious anti-tumor activity through verification of a mouse tumor model.
Term(s) for
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 "nanobody of the present invention", "PD-1 antibody", "PD-1 nanobody" have the same meaning and all refer to nanobodies that specifically recognize and bind to PD-1 (including human PD-1). Preferably, the variable region of the nanobody of the present invention has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3. More preferably, the framework region of the nanobody of the present invention has (a) FR1 shown by SEQ ID NO. 4, FR2 shown by SEQ ID NO. 5, FR3 shown by SEQ ID NO. 6, and FR4 shown by SEQ ID NO. 7, or (b) FR1 shown by SEQ ID NO. 10, FR2 shown by SEQ ID NO. 11, FR3 shown by SEQ ID NO. 12, and FR4 shown by SEQ ID NO. 13.
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 "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 complete 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 nanobody of the present invention can be evaluated or determined by KD values (dissociation constants) (or other means of determination), such as biofilm layer interference technique (Bio-layer interference BLI), measured using FortebioRed96 instrument.
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 comprises a cell surface marker or antigen combined with the anti-PD-1 nano antibody or the 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 present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to a polypeptide that specifically binds to a PD-1 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 polypeptide having a binding activity to PD-1 protein, which includes the above-mentioned CDR region. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. 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 nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present 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.
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-1 protein molecules, thus being used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10mg/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 nanobodies
In a preferred embodiment of the invention, the nanobody 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 present invention, the anti-PD-1 nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.
The anti-PD-1 nano antibody has good specificity and high titer.
Detection method
The invention also relates to a method for detecting PD-1 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-1 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 PD-1 level, which comprises an antibody for identifying the PD-1 protein, a lysis medium for dissolving a sample, and general reagents and buffers required by 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 nanobody of the present invention has wide biological and clinical application values, and its applications relate to the diagnosis and treatment of diseases related to PD-1, research in basic medicine, biological research, etc. One preferred application is for clinical diagnosis and targeted therapy against PD-1, such as tumor therapy.
The main advantages of the invention include:
(a) the nano antibody is specific to human PD-1 protein with a correct spatial structure.
(b) The nano antibody has good blocking activity of PD-1/PD-L1.
(c) The nano antibody has obvious anti-tumor activity.
(d) The production of the nano antibody is simple and convenient.
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: expression and purification of human PD-1 protein
The expression and purification method of the human PD-1 protein comprises the following steps:
(1) synthesizing the nucleotide sequence of human PD-1 on a pCDNA3.1(-) vector, and then subcloning the extracellular segment sequence of the pCDNA3.1(-) vector to a pFUSE-IgG1 vector; (2) extracting the constructed pFUSE-IgG1-hPD-1(ECD) plasmid by using an Omega plasmid large-extract kit; (3) Culturing HEK293F cells to OD 2.0X 106Per mL; (4) mixing the plasmid and the PEI 1:3 evenly, standing for 20min, adding into HEK293F cells at 37 ℃ and 6% CO2Culturing in a shaking incubator for 5-6 days; (5) collecting cell supernatant, and combining with Protein A beads at room temperature for 1 h; (6) after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M Glycine at pH 3.0; (7) eluted proteins were ultrafiltered into PBS and samples were taken for SDS-PAGE detection after yield determination.
The detection result is shown in figure 1, the purity of the protein hPD-1(ECD) -Fc reaches more than 90%, and the residual protein is stored in a refrigerator at the temperature of 80 ℃ below zero for later use.
Example 2: construction and screening of PD-1 nano antibody library
The library construction method is as follows:
(1) mixing 1mg hPD-1(ECD) -Fc antigen with Freund's adjuvant in equal volume, immunizing a Xinjiang bactrian camel once a week for 7 times, and stimulating B cells to express antigen-specific nano-antibody; (2) after 7 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA; (3) synthesizing cDNA and amplifying VHH by using nested PCR; (4) mu.g of pMECS phage display vector (supplied by Biovector) and 10. mu.g of VHH were digested with restriction enzymes Pst I and Not I and the two fragments were ligated; (5) the ligation products were transformed into electroporation competent cells TG1, and a PD-1 nanobody library was constructed and the library volume was determined.
As a result, as shown in FIG. 2, the size of the storage capacity was 2.2X 109CFU。
And randomly picking 24 clones for colony PCR detection, wherein the result shows that the insertion rate of the constructed library is 95.8%.
The antibody screening and identifying method comprises the following steps:
(1) the solution was dissolved in 100mM NaHCO 310 μ g of hPD-1(ECD) -Fc antigen (10 μ g of Fc in NaHCO) at pH 8.23As a control) were coupled to NUNC enzyme plates and left overnight at 4 ℃; (2) adding 100 μ L of 0.1% BSA the next day, blocking for 2h at room temperature; (3) after 2h, 100. mu.L phage (2X 10) was added11CFU immune camel nanometer antibody phage display gene library) and acting for 1h at room temperature; (4) wash 5 times with 0.05% PBS + Tween-20 to wash away non-specsA heterologous bacteriophage; (5) phage specifically binding to PD-1 were dissociated with 100mM triethanolamine and infected with E.coli TG1 cells grown in log phase, cultured at 37 ℃ for 1h, phage generated and purified for the next round of screening, and the same screening process was repeated for 6 rounds to enrich positive clones. (6) From the cell culture dish containing the phage after enrichment, 200 single colonies were picked and inoculated in TB medium containing 100. mu.g/mL ampicillin (2.3 g KH in 1L TB medium)2PO4，12.52g K2HPO412g peptone, 24g yeast extract, 4mL glycerol, after growth to logarithmic phase, add 1mM final concentration of IPTG and incubate overnight at 28 ℃. (7) Crude antibody was obtained by permeation and transferred to an antigen coated ELISA plate and left at room temperature for 1 h. (8) Unbound antibody was washed away with PBST, and a murine anti-HA antibody (COVENCE) was added and left at room temperature for 1 h. (9) Unbound antibody was washed away with PBST, and goat anti-mouse alkaline phosphatase-labeled antibody was added and left at room temperature for 1 h. (10) Unbound antibody was washed away with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution. (11) When the OD value of the sample well is more than 3 times larger than that of the control well (Ratio +/- > 3), the sample well is judged to be a positive clone well. (12) Bacteria from positive clonal wells were shaken in LB liquid containing 100. mu.g/mL Amp to extract plasmids and sequenced.
The enrichment result of the positive clone is shown in fig. 3, and the result shows that the PD-1 nanometer antibody phage is enriched by 21.3 times through six rounds of phage display screening.
And storing the antibody strains with different sequencing results, and screening the functional antibodies.
Example 3: construction and identification of PD-1 overexpression cell line
The construction and identification method comprises the following steps:
(1) reviving CHOZn cells in CO2Incubator overnight (5% CO)2At 37 ℃ C. (2) The packaging plasmid and the target plasmid are mixed with the transfection reagent according to a proper proportion and then added into the CHOZn cell. (3) Viral supernatants were collected at 48 and 72 hours post-transfection, respectively. (4) Using concentrated reagents and virusesMixing the clear liquid according to a certain proportion, uniformly mixing the clear liquid once every half hour, and storing the mixture at 4 ℃ overnight. (5) Centrifuging at 4000rpm for half an hour, removing the supernatant, resuspending the pellet with the target medium and adding to CHOZn cells. (6) After 48 hours, puromycin of appropriate concentration was added for screening. (7) The puromycin-containing fresh medium was changed every other day or every two days depending on the growth of the cells until few cells continued to die. (8) CHOZn cells before and after construction are respectively taken, are stained for proper time by hPD-L1-biotin, then are stained for certain time by SA-PE, and finally are detected by a BD Calibur flow cytometer.
As shown in FIG. 4, CHOZn cells before construction did not bind to hPD-L1-biotin at low concentration; the constructed CHOZn/PD-1 cell can be combined with PD-L1-biotin, which shows that the constructed CHOZn cell over-expressed by PD-1 is successful and can be used for the subsequent screening of PD-1 nano antibody.
Example 4: blocking type PD-1 nano antibody cell level primary screen
The primary screening method comprises the following steps:
(1) inoculating different clone strains with correct sequencing into 1ml of TB medium containing ampicillin with proper concentration, carrying out shake culture at constant temperature of 37 ℃ until logarithmic phase, adding IPTG inducer, and carrying out induction for 16h at 28 ℃; (2) after 16h, breaking the thalli by using an osmotic pressure impact method to obtain a crude extract of the nano antibody; (3) taking 1X 10 of each sample6Suspending CHOZn/PD-1 cells in 0.5% BSA-PBS buffer, respectively adding 200 μ L of the PD-1 nano antibody crude extract, setting a negative control (GFP Nb), adding hPD-L1-Biotin with a proper concentration into all samples, and incubating for 20min at 4 ℃; (5) washing with 1 XPBS for 2 times, adding SA-PE, incubating at 4 ℃ in dark for 20min, washing with 1 XPBS for 2 times, and detecting with flow cytometer (BD Caliber).
The result is shown in fig. 5, a PD-1 nm antibody with good blocking effect is preliminarily screened.
Example 5: expression and purification of PD-1 nano antibody in eukaryotic cell HEK293 and flow cytometry detection of blocking function of nano antibody
The method for expressing the PD-1Nb-Fc fusion protein in the eukaryotic cell HEK293F is as follows:
(1) the PD-1Nb sequence with correct sequencing result is cloned to a pFUSE-IgG4 vector (purchased from Invivogen), and the plasmid is extracted by an Omega plasmid macroextraction kit; (2) culturing HEK293F cells to OD 2.0X 106Per mL; (3) mixing the plasmid and PEI as transfection reagent at a ratio of 1:3, standing for 20min, adding into HEK293F cell at 37 deg.C and 6% CO2Culturing in a shaking incubator for 5-6 days; (4) collecting cell supernatant, and combining with Protein A beads at room temperature for 1 h; (5) after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M Glycine pH 3.0; (6) the eluted proteins were ultrafiltered into PBS and samples were taken after yield determination for SDS-PAGE detection
The results show that the PD-1Nb-Fc fusion protein is successfully expressed in the eukaryotic cell HEK293F, and the residual protein is stored in a refrigerator at-80 ℃ for later use.
The method for identifying the blocking function of the nano antibody by flow cytometry comprises the following steps:
(1) taking 5X 10 samples of each5PD-1 stable cells CHOZn/PD-1 in 0.5% BSA-PBS buffer, adding gradient diluted PD-1Nb, and control antibody Keytruda (antibody dilution gradient 40ug/ml, 20ug/ml, 10ug/m, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 0.625ug/ml, 0.3125ug/ml, 0.1563ug/ml, 0.07813ug/ml, 0.0391ug/ml), adding 100uL of each sample, adding 50ug/ml hPD-L1-Biotin to all samples at the same time, and incubating at 4 deg.C for 20 min; (2) the cells were washed 2 times with PBS, SA-PE was added, incubated at 4 ℃ for 20min, and the cells were washed 2 times with PBS and then examined with a flow cytometer (BD Calibur), and data was processed using graphpad prism 6 software.
The results are shown in FIG. 6, IC of PD-1Nb501.305ug/mL, and IC of the control antibody Keytruda50It was 1.339 g/mL. The blocking activity of the PD-1 nano antibody is similar to that of a control antibody.
Example 6: analysis of cell-level binding Activity of PD-1 Nanobody
The analysis method is as follows:
(1) taking 5X 10 samples of each5CHOZn/PD-1 cells were stably transfected in 0.5% BSA-PBS buffer, and a gradient dilution of PD-1Nb was added, along with a control antibody Keytruda (antibody dilution gradient 160ug/ml, 80ug/ml, 40ug/ml, 20ug/ml, 10 ug/ml)ug/m, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 0.625ug/ml, 0.3125ug/ml, 0.1563ug/ml, 0.0781ug/ml), adding 100uL of each sample, and incubating at 4 deg.C for 20 min; (2) the cells were washed 2 times with PBS, anti Human IgG-FITC was added, incubated at 4 ℃ for 20min, and the cells were washed 2 times with PBS and then detected with a flow cytometer (BD Calibur) and processed with the graphpad prism 6 software.
The results are shown in FIG. 7, EC for PD-1Nb501.064ug/mL, EC of the control antibody Keytruda50It was 1.113 ug/mL.
Example 7: humanized engineering of PD-1 nanobodies
Firstly, taking a PD-1 nano antibody sequence shown in SEQ ID NO. 8 as a template to search a homologous structure in a structure database, and taking a structure of which the E value is 0.0 and the sequence identity is more than or equal to 70%; secondly, comparing the structures, finally selecting the protein including 3dwt according to the resolution of the crystal structure and the constructed evolutionary tree, performing multi-template homologous modeling based on the PD-1 nano antibody sequence shown in SEQ ID NO. 8, selecting the structure with the lowest molpdf according to the high-low order of the scoring function, and continuing the following work; then for the optimal structure of the model, calculating the solvent accessibility of the residues by using a ProtSA server, namely taking the ratio of the folded state of the residues to the solvent contactable area of the unfolded state as a criterion, and taking more than 40 percent of the residues as the residues exposed outside the solvent; finally, the modeled optimal structure and DP-47 were aligned to replace the corresponding solvent-exposed residues.
Finally, a humanized PD-1 nano antibody is obtained by determination, and is coded by an amino acid sequence shown as SEQ ID NO. 14. The antibody sequences before and after humanization correspond to table 1 below:
TABLE 1
Figure BDA0002664765650000201
Example 8: analysis of cell-level blocking activity of humanized PD-1 nanobody (HuNb-Fc)
The humanized nanobody prepared in example 7 was fused with human Fc protein, and the HuNb-Fc amino acid sequence (SEQ ID NO:16) was synthesized into pCDNA3.1(-) vector according to the base sequence (SEQ ID NO:17) optimized for human codons, and then the HuNb-Fc antibody was purified by expression according to the method in example 1. The purified antibody was used in an activity detection assay.
The specific procedure is as in example 5.
The results are shown in FIG. 8, IC at the cellular level of HuNb-Fc501.169ug/ml, similar to the blocking activity of the control antibody Ketytruda.
Example 9: humanized PD-1 nano antibody (HuNb-Fc) PD-1/PD-L1 reporter gene analysis
The analysis method is as follows
(1) Digesting GS-C2/PD-L1 cells for 3-5min by using Accutase enzyme, neutralizing the Accutase enzyme by using F12K complete culture medium, centrifuging at 800rpm for 5min, removing supernatant, re-suspending by using 2mL of F12K complete culture medium, counting, taking corresponding volume to a cell loading slot, and finally obtaining the cell loading slot with the concentration of 5 multiplied by 105Mixing well, transferring the well-mixed cell solution to a corresponding 384-well plate by a discharging gun at 37 ℃ and 5% CO2Incubate overnight. (2) The next day, the antibody samples were diluted. GS-C2/PD-L1 cell culture medium was aspirated away, and the diluted and mixed antibody samples were transferred to the corresponding 384-well plates using a line gun. (3) The number of GS-J2/PD-1 cells required for the experiment was calculated. Centrifuging GS-J2/PD-1 cells, discarding supernatant, resuspending with 1% Assay buffer, transferring the well-mixed cell solution to corresponding 384-well plate, 37 ℃, and 5% CO2Incubate for 6 h. (4) Reading a plate: firstly, taking the appropriate volume of ONE-GloTMAnd (3) adding the solution of the Luciferase Assay System into a sample adding groove, incubating for 5-10min at room temperature in a dark place at 30 ul/hole, and adjusting a BioTeK machine to a bioluminescence mode for detection.
As shown in FIG. 9, HuNb-Fc has similar biological activity to Keytruda.
Example 10: construction and activity analysis of humanized multivalent PD1 nano antibody
In order to further improve the blocking activity of the humanized antibody, an activity analysis was performed after attempting to construct a multivalent antibody, and the structure thereof is shown below:
a: HuNb-L1-HuNb-Fc (amino acid sequence shown in SEQ ID NO:18, base sequence shown in SEQ ID NO:21),
b: HuNb-L2-HuNb-Fc (the amino acid sequence is shown in SEQ ID NO:19, and the base sequence is shown in SEQ ID NO:22),
c: HuNb-L3-HuNb-Fc (amino acid sequence shown in SEQ ID NO:20, base sequence shown in SEQ ID NO:23),
d: HuNb-L1-HuNb-L1-HuNb-Fc (the amino acid sequence is shown in SEQ ID NO:24, and the base sequence is shown in SEQ ID NO:27),
e: HuNb-L2-HuNb-L2-HuNb-Fc (amino acid sequence is shown in SEQ ID NO:25, base sequence is shown in SEQ ID NO:28),
f: HuNb-L3-HuNb-L3-HuNb-Fc (the amino acid sequence is shown in SEQ ID NO:26, and the base sequence is shown in SEQ ID NO:29),
wherein HuNb refers to the humanized PD1 nanobody prepared in example 7, L1 refers to linker GS, L2 refers to linker (4GS)2L3 denotes a linker (4GS)4。
The base sequences of the above-form multivalent antibodies were synthesized onto pcdna3.1(-) vectors, and then each antibody was expressed and purified using the method in example 1, and then the blocking activity analysis was performed using the detection method in example 9.
The results are shown in FIG. 10: the activity of the multivalent antibody is obviously improved compared with that of a bivalent antibody HuNb-Fc (SEQ ID NO:16), wherein the blocking activity of the 4-valent antibody with the C structure and the 6-valent antibody with the E structure is better, and the antibody with the C structure is selected for further verification.
Example 11: in vivo efficacy research of humanized PD-1 nano antibody
hPD-1 transgenic mice were inoculated with MC38 cells and divided into three groups after tumor formation, and 8 of them were administered to each group (negative control group: PBS; positive control group: Keytruda; experimental group: antibody C prepared in example 10. administration frequency: 1 every three days, 7 times of continuous administration: administration dose: 10 mg/kg).
The results of the experiment are shown in FIG. 11: antibody C showed significant tumor suppression with a TGI of 91%, and complete tumor regression in one mouse, which was significantly better than that of Keytruda (TGI of 60%. FIG. 12 shows the results of tumor-stripping weighing after mice became sacrificed, and it can be seen that the tumor weight of mice in antibody C group was significantly lower than that of Keytruda group and negative control group.
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> blocking type PD-1 nano antibody and coding sequence and application thereof
<130> P2020-1599
<150> CN201910833395.6
<151> 2019-09-04
<160> 29
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ggccagggca ccctggtgac cgtgagcagc gagagcaagt acggcccccc ctgccccccc 420
tgccccgccc ccgagttcct gggcggcccc agcgtgttcc tgttcccccc caagcccaag 480
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cccagcagca tcgagaagac catcagcaag gccaagggcc agcccaggga gccccaggtg 780
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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
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Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
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Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
485 490
<210> 19
<211> 498
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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 Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro
100 105 110
Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
115 120 125
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg
180 185 190
Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
195 200 205
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
210 215 220
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu
225 230 235 240
Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr
245 250 255
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
275 280 285
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
290 295 300
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
305 310 315 320
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
325 330 335
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
340 345 350
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
355 360 365
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
370 375 380
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
385 390 395 400
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
405 410 415
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
420 425 430
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
435 440 445
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
450 455 460
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
465 470 475 480
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
485 490 495
Gly Lys
<210> 20
<211> 509
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Glu 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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 Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro
100 105 110
Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
115 120 125
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Gly Gly
145 150 155 160
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
165 170 175
Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly
180 185 190
Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser
195 200 205
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
210 215 220
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu Ser Gly Tyr Tyr Ser
245 250 255
Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln
260 265 270
Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
275 280 285
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
290 295 300
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
305 310 315 320
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
325 330 335
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
340 345 350
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
355 360 365
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
370 375 380
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
385 390 395 400
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
405 410 415
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
420 425 430
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
435 440 445
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
450 455 460
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
465 470 475 480
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
485 490 495
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
500 505
<210> 21
<211> 1473
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcagcgagg tgcagctgca ggagagcggc 420
ggcggcctgg tgcagcccgg cggcagcctg aggctgagct gcgccgccag cggctacacc 480
tacagcagct actacatggg ctggttcagg caggcccccg gcaagggcct ggagggcgtg 540
gccgccatcg acagggacgg caggaccagc tacgccgaca gcgtgaaggg caggttcacc 600
atcagcaggg acaacgccaa gaacaccctg tacctgcaga tgaacagcct gagggccgag 660
gacaccgccg tgtactactg cgccgccgtg ctggtgctga gcggctacta cagcgactac 720
ggcctggtgc cccccccctt cgagtacaac tactggggcc agggcaccct ggtgaccgtg 780
agcagcgaga gcaagtacgg ccccccctgc cccccctgcc ccgcccccga gttcctgggc 840
ggccccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 900
cccgaggtga cctgcgtggt ggtggacgtg agccaggagg accccgaggt gcagttcaac 960
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccaggga ggagcagttc 1020
aacagcacct acagggtggt gagcgtgctg accgtgctgc accaggactg gctgaacggc 1080
aaggagtaca agtgcaaggt gagcaacaag ggcctgccca gcagcatcga gaagaccatc 1140
agcaaggcca agggccagcc cagggagccc caggtgtaca ccctgccccc cagccaggag 1200
gagatgacca agaaccaggt gagcctgacc tgcctggtga agggcttcta ccccagcgac 1260
atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac cacccccccc 1320
gtgctggaca gcgacggcag cttcttcctg tacagcaggc tgaccgtgga caagagcagg 1380
tggcaggagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1440
acccagaaga gcctgagcct gagcctgggc aag 1473
<210> 22
<211> 1494
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcggcggcg gcagcggcgg cggcagcgag 420
gtgcagctgc aggagagcgg cggcggcctg gtgcagcccg gcggcagcct gaggctgagc 480
tgcgccgcca gcggctacac ctacagcagc tactacatgg gctggttcag gcaggccccc 540
ggcaagggcc tggagggcgt ggccgccatc gacagggacg gcaggaccag ctacgccgac 600
agcgtgaagg gcaggttcac catcagcagg gacaacgcca agaacaccct gtacctgcag 660
atgaacagcc tgagggccga ggacaccgcc gtgtactact gcgccgccgt gctggtgctg 720
agcggctact acagcgacta cggcctggtg ccccccccct tcgagtacaa ctactggggc 780
cagggcaccc tggtgaccgt gagcagcgag agcaagtacg gccccccctg ccccccctgc 840
cccgcccccg agttcctggg cggccccagc gtgttcctgt tcccccccaa gcccaaggac 900
accctgatga tcagcaggac ccccgaggtg acctgcgtgg tggtggacgt gagccaggag 960
gaccccgagg tgcagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc 1020
aagcccaggg aggagcagtt caacagcacc tacagggtgg tgagcgtgct gaccgtgctg 1080
caccaggact ggctgaacgg caaggagtac aagtgcaagg tgagcaacaa gggcctgccc 1140
agcagcatcg agaagaccat cagcaaggcc aagggccagc ccagggagcc ccaggtgtac 1200
accctgcccc ccagccagga ggagatgacc aagaaccagg tgagcctgac ctgcctggtg 1260
aagggcttct accccagcga catcgccgtg gagtgggaga gcaacggcca gcccgagaac 1320
aactacaaga ccaccccccc cgtgctggac agcgacggca gcttcttcct gtacagcagg 1380
ctgaccgtgg acaagagcag gtggcaggag ggcaacgtgt tcagctgcag cgtgatgcac 1440
gaggccctgc acaaccacta cacccagaag agcctgagcc tgagcctggg caag 1494
<210> 23
<211> 1527
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcggcggcg gcagcggcgg cggcggcagc 420
ggcggcggcg gcagcggcgg cggcggcagc gaggtgcagc tgcaggagag cggcggcggc 480
ctggtgcagc ccggcggcag cctgaggctg agctgcgccg ccagcggcta cacctacagc 540
agctactaca tgggctggtt caggcaggcc cccggcaagg gcctggaggg cgtggccgcc 600
atcgacaggg acggcaggac cagctacgcc gacagcgtga agggcaggtt caccatcagc 660
agggacaacg ccaagaacac cctgtacctg cagatgaaca gcctgagggc cgaggacacc 720
gccgtgtact actgcgccgc cgtgctggtg ctgagcggct actacagcga ctacggcctg 780
gtgccccccc ccttcgagta caactactgg ggccagggca ccctggtgac cgtgagcagc 840
gagagcaagt acggcccccc ctgccccccc tgccccgccc ccgagttcct gggcggcccc 900
agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag gacccccgag 960
gtgacctgcg tggtggtgga cgtgagccag gaggaccccg aggtgcagtt caactggtac 1020
gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gttcaacagc 1080
acctacaggg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1140
tacaagtgca aggtgagcaa caagggcctg cccagcagca tcgagaagac catcagcaag 1200
gccaagggcc agcccaggga gccccaggtg tacaccctgc cccccagcca ggaggagatg 1260
accaagaacc aggtgagcct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 1320
gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1380
gacagcgacg gcagcttctt cctgtacagc aggctgaccg tggacaagag caggtggcag 1440
gagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1500
aagagcctga gcctgagcct gggcaag 1527
<210> 24
<211> 623
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Glu 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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 Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro
100 105 110
Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
115 120 125
Ser Ser Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
130 135 140
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
145 150 155 160
Tyr Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly
165 170 175
Leu Glu Gly Val Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala
180 185 190
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
195 200 205
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
210 215 220
Tyr Tyr Cys Ala Ala Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr
225 230 235 240
Gly Leu Val Pro Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr
245 250 255
Leu Val Thr Val Ser Ser Gly Ser Glu Val Gln Leu Gln Glu Ser Gly
260 265 270
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
275 280 285
Ser Gly Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala
290 295 300
Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg Asp Gly Arg
305 310 315 320
Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
325 330 335
Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
340 345 350
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu Ser Gly Tyr
355 360 365
Tyr Ser Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr Asn Tyr Trp
370 375 380
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro
385 390 395 400
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
405 410 415
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
420 425 430
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
435 440 445
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
450 455 460
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
465 470 475 480
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
485 490 495
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
500 505 510
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
515 520 525
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
530 535 540
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
545 550 555 560
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
565 570 575
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
580 585 590
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
595 600 605
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
610 615 620
<210> 25
<211> 637
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Glu 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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 Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro
100 105 110
Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
115 120 125
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg
180 185 190
Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
195 200 205
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
210 215 220
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu
225 230 235 240
Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr
245 250 255
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Gly Gly
275 280 285
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
290 295 300
Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly
305 310 315 320
Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser
325 330 335
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
340 345 350
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
355 360 365
Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu Ser Gly Tyr Tyr Ser
370 375 380
Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln
385 390 395 400
Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys
405 410 415
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
420 425 430
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
435 440 445
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
450 455 460
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
465 470 475 480
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
485 490 495
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
500 505 510
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
515 520 525
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
530 535 540
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
545 550 555 560
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
565 570 575
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
580 585 590
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
595 600 605
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
610 615 620
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
625 630 635
<210> 26
<211> 659
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Glu 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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 Val Leu Val Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro
100 105 110
Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
115 120 125
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Gly Gly
145 150 155 160
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
165 170 175
Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly
180 185 190
Lys Gly Leu Glu Gly Val Ala Ala Ile Asp Arg Asp Gly Arg Thr Ser
195 200 205
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
210 215 220
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Ala Ala Val Leu Val Leu Ser Gly Tyr Tyr Ser
245 250 255
Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu Tyr Asn Tyr Trp Gly Gln
260 265 270
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
290 295 300
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
305 310 315 320
Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly Trp
325 330 335
Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala Ile Asp
340 345 350
Arg Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
355 360 365
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
370 375 380
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Leu Val
385 390 395 400
Leu Ser Gly Tyr Tyr Ser Asp Tyr Gly Leu Val Pro Pro Pro Phe Glu
405 410 415
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser
420 425 430
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
435 440 445
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
450 455 460
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
465 470 475 480
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
485 490 495
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
500 505 510
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
515 520 525
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
530 535 540
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
545 550 555 560
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
565 570 575
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
580 585 590
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
595 600 605
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
610 615 620
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
625 630 635 640
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
645 650 655
Leu Gly Lys
<210> 27
<211> 1869
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcagcgagg tgcagctgca ggagagcggc 420
ggcggcctgg tgcagcccgg cggcagcctg aggctgagct gcgccgccag cggctacacc 480
tacagcagct actacatggg ctggttcagg caggcccccg gcaagggcct ggagggcgtg 540
gccgccatcg acagggacgg caggaccagc tacgccgaca gcgtgaaggg caggttcacc 600
atcagcaggg acaacgccaa gaacaccctg tacctgcaga tgaacagcct gagggccgag 660
gacaccgccg tgtactactg cgccgccgtg ctggtgctga gcggctacta cagcgactac 720
ggcctggtgc cccccccctt cgagtacaac tactggggcc agggcaccct ggtgaccgtg 780
agcagcggca gcgaggtgca gctgcaggag agcggcggcg gcctggtgca gcccggcggc 840
agcctgaggc tgagctgcgc cgccagcggc tacacctaca gcagctacta catgggctgg 900
ttcaggcagg cccccggcaa gggcctggag ggcgtggccg ccatcgacag ggacggcagg 960
accagctacg ccgacagcgt gaagggcagg ttcaccatca gcagggacaa cgccaagaac 1020
accctgtacc tgcagatgaa cagcctgagg gccgaggaca ccgccgtgta ctactgcgcc 1080
gccgtgctgg tgctgagcgg ctactacagc gactacggcc tggtgccccc ccccttcgag 1140
tacaactact ggggccaggg caccctggtg accgtgagca gcgagagcaa gtacggcccc 1200
ccctgccccc cctgccccgc ccccgagttc ctgggcggcc ccagcgtgtt cctgttcccc 1260
cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg 1320
gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt acgtggacgg cgtggaggtg 1380
cacaacgcca agaccaagcc cagggaggag cagttcaaca gcacctacag ggtggtgagc 1440
gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgagc 1500
aacaagggcc tgcccagcag catcgagaag accatcagca aggccaaggg ccagcccagg 1560
gagccccagg tgtacaccct gccccccagc caggaggaga tgaccaagaa ccaggtgagc 1620
ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 1680
ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga cggcagcttc 1740
ttcctgtaca gcaggctgac cgtggacaag agcaggtggc aggagggcaa cgtgttcagc 1800
tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgagc 1860
ctgggcaag 1869
<210> 28
<211> 1911
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcggcggcg gcagcggcgg cggcagcgag 420
gtgcagctgc aggagagcgg cggcggcctg gtgcagcccg gcggcagcct gaggctgagc 480
tgcgccgcca gcggctacac ctacagcagc tactacatgg gctggttcag gcaggccccc 540
ggcaagggcc tggagggcgt ggccgccatc gacagggacg gcaggaccag ctacgccgac 600
agcgtgaagg gcaggttcac catcagcagg gacaacgcca agaacaccct gtacctgcag 660
atgaacagcc tgagggccga ggacaccgcc gtgtactact gcgccgccgt gctggtgctg 720
agcggctact acagcgacta cggcctggtg ccccccccct tcgagtacaa ctactggggc 780
cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cagcgaggtg 840
cagctgcagg agagcggcgg cggcctggtg cagcccggcg gcagcctgag gctgagctgc 900
gccgccagcg gctacaccta cagcagctac tacatgggct ggttcaggca ggcccccggc 960
aagggcctgg agggcgtggc cgccatcgac agggacggca ggaccagcta cgccgacagc 1020
gtgaagggca ggttcaccat cagcagggac aacgccaaga acaccctgta cctgcagatg 1080
aacagcctga gggccgagga caccgccgtg tactactgcg ccgccgtgct ggtgctgagc 1140
ggctactaca gcgactacgg cctggtgccc ccccccttcg agtacaacta ctggggccag 1200
ggcaccctgg tgaccgtgag cagcgagagc aagtacggcc ccccctgccc cccctgcccc 1260
gcccccgagt tcctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc 1320
ctgatgatca gcaggacccc cgaggtgacc tgcgtggtgg tggacgtgag ccaggaggac 1380
cccgaggtgc agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 1440
cccagggagg agcagttcaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 1500
caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggg cctgcccagc 1560
agcatcgaga agaccatcag caaggccaag ggccagccca gggagcccca ggtgtacacc 1620
ctgcccccca gccaggagga gatgaccaag aaccaggtga gcctgacctg cctggtgaag 1680
ggcttctacc ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac 1740
tacaagacca ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaggctg 1800
accgtggaca agagcaggtg gcaggagggc aacgtgttca gctgcagcgt gatgcacgag 1860
gccctgcaca accactacac ccagaagagc ctgagcctga gcctgggcaa g 1911
<210> 29
<211> 1977
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 300
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 360
ggccagggca ccctggtgac cgtgagcagc ggcggcggcg gcagcggcgg cggcggcagc 420
ggcggcggcg gcagcggcgg cggcggcagc gaggtgcagc tgcaggagag cggcggcggc 480
ctggtgcagc ccggcggcag cctgaggctg agctgcgccg ccagcggcta cacctacagc 540
agctactaca tgggctggtt caggcaggcc cccggcaagg gcctggaggg cgtggccgcc 600
atcgacaggg acggcaggac cagctacgcc gacagcgtga agggcaggtt caccatcagc 660
agggacaacg ccaagaacac cctgtacctg cagatgaaca gcctgagggc cgaggacacc 720
gccgtgtact actgcgccgc cgtgctggtg ctgagcggct actacagcga ctacggcctg 780
gtgccccccc ccttcgagta caactactgg ggccagggca ccctggtgac cgtgagcagc 840
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 900
gaggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 960
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggtt caggcaggcc 1020
cccggcaagg gcctggaggg cgtggccgcc atcgacaggg acggcaggac cagctacgcc 1080
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 1140
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgtgctggtg 1200
ctgagcggct actacagcga ctacggcctg gtgccccccc ccttcgagta caactactgg 1260
ggccagggca ccctggtgac cgtgagcagc gagagcaagt acggcccccc ctgccccccc 1320
tgccccgccc ccgagttcct gggcggcccc agcgtgttcc tgttcccccc caagcccaag 1380
gacaccctga tgatcagcag gacccccgag gtgacctgcg tggtggtgga cgtgagccag 1440
gaggaccccg aggtgcagtt caactggtac gtggacggcg tggaggtgca caacgccaag 1500
accaagccca gggaggagca gttcaacagc acctacaggg tggtgagcgt gctgaccgtg 1560
ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtgagcaa caagggcctg 1620
cccagcagca tcgagaagac catcagcaag gccaagggcc agcccaggga gccccaggtg 1680
tacaccctgc cccccagcca ggaggagatg accaagaacc aggtgagcct gacctgcctg 1740
gtgaagggct tctaccccag cgacatcgcc gtggagtggg agagcaacgg ccagcccgag 1800
aacaactaca agaccacccc ccccgtgctg gacagcgacg gcagcttctt cctgtacagc 1860
aggctgaccg tggacaagag caggtggcag gagggcaacg tgttcagctg cagcgtgatg 1920
cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagcct gggcaag 1977

Claims (21)

1. A VHH chain of an anti-PD-1 nanobody, characterized in that the VHH chain is composed of a framework region FR and a complementarity determining region CDR, wherein the CDR is CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
2. The VHH chain according to claim 1, wherein the framework regions FR are:
(a) FR1 shown by SEQ ID NO. 4, FR2 shown by SEQ ID NO. 5, FR3 shown by SEQ ID NO. 6, and FR4 shown by SEQ ID NO. 7; or
(b) FR1 shown in SEQ ID NO. 10, FR2 shown in SEQ ID NO. 11, FR3 shown in SEQ ID NO. 12, and FR4 shown in SEQ ID NO. 13.
3. An anti-PD-1 nanobody, which is a nanobody against a PD-1 epitope and has a VHH chain of an amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 14.
4. An anti-PD-1 multivalent antibody, characterized in that said multivalent antibody comprises 2, 3 or 4 VHH chains of the anti-PD-1 nanobody of claim 1 or the anti-PD-1 nanobody of claim 3.
5. The multivalent antibody of claim 4, wherein the multivalent antibody has a structure from N-terminus to C-terminus of formula II or formula III:
P-L' -P-Fc formula II
P-L '-P-L' -P-Fc formula III
Wherein,
"-" is a peptide bond;
l' is a component of the joint,
p is an anti-PD-1 nano antibody,
fc is the Fc segment of the antibody.
6. The multivalent antibody of claim 4, wherein the multivalent antibody has an amino acid sequence as set forth in SEQ ID NOS 18-20 and 24-26.
7. The multivalent antibody of claim 4, wherein the multivalent antibody can form a dimer.
8. A bispecific antibody, wherein said bispecific antibody comprises: an anti-PD-1 nanobody and a second antibody, wherein the Complementarity Determining Regions (CDRs) of the anti-PD-1 nanobody are:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3.
9. The bispecific antibody of claim 8, wherein the second antibody comprises: 4-1BB nanobody, CD47 nanobody, VEGF nanobody, HER2 nanobody, EGFR nanobody, HER3 nanobody, B7H3 nanobody, TIGIT nanobody, OX-40 nanobody, CD40 nanobody or PD-L1 nanobody.
10. A polynucleotide encoding a protein selected from the group consisting of: the VHH chain of the anti-PD-1 nanobody of claim 1, the anti-PD-1 nanobody of claim 3, the anti-PD-1 multivalent antibody of claim 4, or the bispecific antibody of claim 8.
11. The polynucleotide of claim 10, wherein the polynucleotide has the sequence shown in SEQ ID No.9 or 15.
12. An expression vector comprising the polynucleotide of claim 10.
13. A host cell comprising the expression vector of claim 12, or having the polynucleotide of claim 10 integrated into its genome,
or the host cell expresses the anti-PD-1 nanobody of claim 3, the anti-PD-1 multivalent antibody of claim 4, or the bispecific antibody of claim 8.
14. A method of producing an anti-PD-1 antibody, comprising the steps of: (a) culturing the host cell of claim 13 under conditions suitable for production of an antibody, thereby obtaining a culture comprising the anti-PD-1 antibody; and (b) isolating or recovering the anti-PD-1 antibody from the culture.
15. An immunoconjugate, comprising: (a) the VHH chain of the anti-PD-1 nanobody of claim 1, the anti-PD-1 nanobody of claim 3, the anti-PD-1 multivalent antibody of claim 4, or the bispecific antibody of claim 8; and (b) a coupling moiety selected from the group consisting of: a detectable label or drug.
16. The immunoconjugate of claim 15, wherein the drug is a toxin, a cytokine, or an enzyme.
17. Use of the anti-PD-1 nanobody of claim 3, the anti-PD-1 multivalent antibody of claim 4 or the immunoconjugate of claim 15, for (a) the preparation of a reagent for the detection of a PD-1 molecule; and/or (b) for the preparation of a medicament for the treatment of tumours.
18. A pharmaceutical composition comprising: (i) the VHH chain of claim 1, the anti-PD-1 nanobody of claim 3, the anti-PD-1 multivalent antibody of claim 4, the bispecific antibody of claim 8 or the immunoconjugate of claim 15; and (ii) a pharmaceutically acceptable carrier.
19. A method for detecting PD-1 protein in a sample, said method comprising the steps of: (1) contacting a sample with the nanobody of claim 3 or the anti-PD-1 multivalent antibody of claim 4; (2) detecting whether an antigen-antibody complex of the nanobody or the anti-PD-1 multivalent antibody with PD-1 protein is formed, wherein the formation of the complex indicates the presence of PD-1 protein in the sample, which is a non-diagnostic, non-therapeutic method.
20. A PD-1 protein detection reagent, wherein said detection reagent comprises the immunoconjugate of claim 15 and a detectably acceptable carrier.
21. Use of the immunoconjugate of claim 15 for the preparation of a contrast agent for the in vivo detection of PD-1 protein.
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