WO2021197358A1 - 一种抗pd-l1和pd-l2抗体及其衍生物和用途 - Google Patents

一种抗pd-l1和pd-l2抗体及其衍生物和用途 Download PDF

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WO2021197358A1
WO2021197358A1 PCT/CN2021/084196 CN2021084196W WO2021197358A1 WO 2021197358 A1 WO2021197358 A1 WO 2021197358A1 CN 2021084196 W CN2021084196 W CN 2021084196W WO 2021197358 A1 WO2021197358 A1 WO 2021197358A1
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nanobody
seq
antibody
amino acid
bispecific antibody
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PCT/CN2021/084196
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English (en)
French (fr)
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缪小牛
吴凡
陈乘
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普米斯生物技术(珠海)有限公司
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Priority to US17/995,197 priority Critical patent/US20230203167A1/en
Priority to EP21780301.4A priority patent/EP4130041A4/en
Priority to CN202180026344.8A priority patent/CN115397857A/zh
Priority to KR1020227037885A priority patent/KR20220160670A/ko
Priority to JP2022559528A priority patent/JP2023519412A/ja
Publication of WO2021197358A1 publication Critical patent/WO2021197358A1/zh

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Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-PD-L1 and PD-L2 antibody and derivatives and uses thereof.
  • PD-1 Programmed death-1
  • CD279 is a member of the CD28 family. Its cytoplasmic region contains two tyrosine residues, and one near the N-terminus is located at the immunoreceptor tyrosine inhibition Among the motifs (immunoreceptor tyrosine-based inhibitory motif, ITIM), one near the C-terminus is located in the immunoreceptor tyrosine-based switch motif (ITSM).
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITIM immunoreceptor tyrosine-based switch motif
  • PD-1 is mainly expressed on the surface of activated T lymphocytes, B lymphocytes and macrophages. Under normal circumstances, PD-1 can inhibit the function of T lymphocytes and promote the function of Treg, thereby inhibiting the autoimmune response and preventing the occurrence of autoimmune diseases.
  • Programmed death factor 1 ligand 1 (programmed death 1 ligand 1, PD-L1), also known as CD274, is a member of the B7 family and a ligand of PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including 1 IgV-like domain, 1 IgC-like domain, 1 transmembrane hydrophobic domain, and 1 intracellular domain composed of 30 amino acids.
  • PD-L1 Different from other B7 family molecules, PD-L1 has the effect of negatively regulating the immune response. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells. In addition to lymphocytes, PD-L1 is also expressed in many other tissues such as thymus, heart, placenta, etc. Endothelial cells, and various non-lymphoid lines such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc.
  • PD-L1 has a certain versatility in regulating autoreactive T and B cells and immune tolerance, and plays a role in T and B cell responses in peripheral tissues.
  • the high expression of PD-L1 on tumor cells is related to the poor prognosis of cancer patients.
  • the combination of PD-L1 expressed by tumor cells and PD-1 can promote the immune escape of tumors through the inhibitory effect on lymphocytes.
  • the combination of PD-L1 and PD-1 can cause a variety of biological changes and cause immune regulation, such as inhibiting the proliferation and activation of lymphocytes, inhibiting the differentiation of CD4+ T cells into Th1 and Th17 cells, and inhibiting the release of inflammatory cytokines Wait.
  • Programmed death factor 1 ligand 2 (PD-L2), also known as CD273 or B7-DC, is another important ligand of PD-1.
  • the interaction of PD-1 and PD-L2 can inhibit the activation and proliferation of CD4+ T cells, reduce the release of cytokines, and increase the immune escape of tumors. Blocking the interaction of PD-1 and PD-L1 and PD-L2 at the same time can effectively liberate the inhibition of the immune system by PD-1 pathway activation, activate the body's own immune system, and kill tumors.
  • single-domain antibodies have high stability and water solubility. It has the characteristics of good sex, simple humanization, high targeting and strong penetrability, and it plays a huge function beyond imagination in immunological experiments, diagnosis and treatment.
  • the purpose of the present invention is to provide a bispecific antibody capable of simultaneously targeting PD-L1 and PD-L2.
  • each complementarity determining region CDR of the VHH chain of the PD-L2 Nanobody consists of the following:
  • the amino acid sequence is CDR1 shown in SEQ ID NO: 57; the amino acid sequence is CDR2 shown in SEQ ID NO: 58; and the amino acid sequence is CDR3 shown in SEQ ID NO: 59; or
  • the amino acid sequence is CDR1 shown in SEQ ID NO: 60; the amino acid sequence is CDR2 shown in SEQ ID NO: 61; and the amino acid sequence is CDR3 shown in SEQ ID NO: 62;
  • amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is shown in SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 17 or 18. .
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is as SEQ ID NO: 3 (ie D-Na-96), 16 (ie HZ-D-Na-96-1) , 12 (ie D-Ye-29) or 15 (ie HZ-D-Ye-29-3).
  • the anti-PD-L2 Nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is as 16 (ie HZ-D-Na-96- 1) or 15 (ie HZ-D-Ye-29-3) as shown.
  • the PD-L2 Nanobody can block the interaction of PD-1 and PD-L2.
  • each complementarity determining region CDR of the VHH chain of the PD-L1 Nanobody is composed of the following:
  • the amino acid sequence is CDR1 shown in SEQ ID NO: 63; the amino acid sequence is CDR2 shown in SEQ ID NO: 64; and the amino acid sequence is CDR3 shown in SEQ ID NO: 65.
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • amino acid sequence of the VHH chain of the anti-PD-L1 Nanobody is as SEQ ID NO: 19 (ie K-Yr-13&14-02), 20 (ie K-Yr-13&14-09) , 21 (ie K-Yr-13&14-16) or 22 (ie HZ-K-Yr-13&14-02-3).
  • the anti-PD-L1 Nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L1 Nanobody is as shown in SEQ ID NO: 22 (ie HZ-K- Yr-13&14-02-3) shown.
  • the PD-L1 Nanobody can block the interaction between PD-1 and PD-L1.
  • a bispecific antibody comprising: the anti-PD-L1 Nanobody as described in the second aspect of the present invention and the anti-PD-L1 Nanobody as described in the first aspect of the present invention Anti-PD-L2 Nanobody.
  • the bispecific antibody includes 1-3 anti-PD-L1 Nanobodies, preferably, includes 1 or 2 anti-PD-L1 Nanobodies.
  • the bispecific antibody includes 1-3 anti-PD-L2 Nanobodies, preferably, includes 1 or 2 anti-PD-L2 Nanobodies.
  • the bispecific antibody further comprises an Fc segment.
  • the Fc segment of the bispecific antibody is selected from the following group: human IgG domain, CH1+CL1 domain, or a combination thereof.
  • the human IgG domain is a modified mutant IgG domain, preferably a LALA mutant IgG domain.
  • the bispecific antibody contains a polypeptide having a structure as shown in formula I or formula II, or a polypeptide having a structure as shown in formula III and formula IV at the same time,
  • a and B are each independently an anti-PD-L1 Nanobody according to the second aspect of the invention or an anti-PD-L2 Nanobody according to the first aspect of the invention, and A and B are different antibodies;
  • L1, L2, L3 and L4 are each independently a peptide bond or linker element
  • Fc1 and Fc2 are the Fc segments of antibodies; wherein Fc1 is a human IgG domain (preferably a LALA mutant IgG domain), and Fc2 is a CH1+CL domain;
  • the bispecific antibody has a polypeptide sequence of the structure shown in formula III and formula IV, and the polypeptide of the structure shown in formula III and the polypeptide of the structure shown in formula IV pass through two The sulfur bond forms a heterodimer a.
  • the bispecific antibody has a polypeptide having the structure shown in Formula I, and the polypeptide forms a homodimer i through the disulfide bond between Fc1.
  • the bispecific antibody has a polypeptide having the structure shown in Formula II, and the polypeptide forms a homodimer ii through the disulfide bond between Fc1.
  • the bispecific antibody has the polypeptide sequence of the structure shown in formula III and formula IV, and the polypeptide of the structure shown in formula III and the polypeptide of the structure shown in formula IV pass through Fc2
  • the disulfide bond between the two forms a heterodimer a
  • the heterodimer ii forms a homodimer iii through the disulfide bond between Fc1.
  • amino acid sequence of the PD-L1 Nanobody is shown in SEQ ID NO: 19, 20, 21 or 22, preferably SEQ ID NO: 22. .
  • the bispecific antibody also includes the VHH chain of other anti-PD-L2 Nanobodies, and the amino acid sequence of the other PD-L2 Nanobodies is as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.
  • amino acid sequence of the VHH chain of the anti-PD-L2 Nanobody is shown in SEQ ID NO: 3, 16, 12 or 15, preferably SEQ ID NO: 16 or 15.
  • the sequence of the linker element is shown in SEQ ID NO: 27, or has a sequence of ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 27 Identity.
  • amino acid sequence of the human IgG domain of the LALA mutant type is shown in SEQ ID NO: 28, or has ⁇ 85% (preferably 90%, more preferably 90%) with the sequence shown in SEQ ID NO: 28. Preferably 95%) sequence identity.
  • the amino acid sequence of the CH1 domain is as shown in SEQ ID NO: 29, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 29 The sequence identity.
  • the amino acid sequence of the CL domain is shown in SEQ ID NO: 30, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 30 The sequence identity.
  • amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 23 or 24.
  • the bispecific antibody contains a polypeptide of the structure shown in formula III and formula IV at the same time, wherein the amino acid sequence of the polypeptide of formula III is shown in SEQ ID NO: 25, The amino acid sequence of the polypeptide described in Formula IV is shown in SEQ ID NO: 26.
  • an isolated polynucleotide encoding the anti-PD-L2 Nanobody as described in the first aspect of the present invention, and the anti-PD-L2 Nanobody as described in the second aspect of the present invention.
  • PD-L1 Nanobody or a bispecific antibody as described in the third aspect of the invention.
  • sequence of the polynucleotide is as SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47 or 48.
  • sequence of the polynucleotide is as SEQ ID NO: 33 (ie D-Na-96), 46 (ie HZ-D-Na-96-1), 42 (ie D-Ye- 29) or 45 (ie HZ-D-Ye-29); preferably SEQ ID NO: 46 or 45.
  • sequence of the polynucleotide is as SEQ ID NO: 49 (i.e. K-Yr-13&14-02), 50 (i.e. K-Yr-13&14-09), 51 (i.e. K-Yr- 13&14-16) or 52 (ie HZ-K-Yr-13&14-02-3); preferably SEQ ID NO: 52.
  • sequence of the polynucleotide is shown in SEQ ID NO: 53 or 54.
  • the polynucleotide includes a first polynucleotide whose sequence is shown in SEQ ID NO: 55 and a second polynucleotide whose sequence is shown in SEQ ID NO: 56.
  • the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vectors include viral vectors, such as Lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a host cell contains the vector according to the fifth aspect of the present invention, or its genome integrates the polynucleotide according to the fourth aspect of the present invention;
  • the host cell expresses the anti-PD-L2 Nanobody according to the first aspect of the invention, the anti-PD-L1 Nanobody according to the second aspect of the invention, or the anti-PD-L1 Nanobody according to the third aspect of the invention Bispecific antibodies.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • a method for producing anti-PD-L1 Nanobody, anti-PD-L2 Nanobody, or bispecific antibody which includes the steps:
  • step (b) Purifying and/or separating the culture obtained in step (a) to obtain the anti-PD-L1 Nanobody, anti-PD-L2 Nanobody, or bispecific antibody.
  • the purification can be purified and separated by a protein A affinity column to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • an immunoconjugate the immunoconjugate containing:
  • a coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combination.
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: 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 a combination thereof; and/or
  • Therapeutic isotope said therapeutic isotope is selected from the following group: 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-133Yb-169, Yb-177, Or a combination.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes (etoposides) taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group:
  • Otostatin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • chlortetracycline mettancilol
  • octoxin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • Lastatin doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissus, dihydroxy anthracisin diketone, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, acacia toxin A chain, capsule lotus root toxin A chain, ⁇ -Sarcina, white tree toxin, mitogellin, retstrictocin, phenomycin, enoxomycin
  • the coupling portion is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles, prodrugs Activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (such as cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (such as bivalent) anti-PD-L2 nanobody as described in the first aspect of the present invention, and an anti-PD-L2 nanobody as described in the second aspect of the present invention.
  • a multivalent (such as bivalent) anti-PD-L2 nanobody as described in the first aspect of the present invention
  • an anti-PD-L2 nanobody as described in the second aspect of the present invention.
  • -L1 Nanobody or a bispecific antibody as described in the third aspect of the invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of the anti-PD-L2 Nanobody as described in the first aspect of the present invention, as in the second aspect of the present invention.
  • the anti-PD-L1 Nanobody according to aspect, or the bispecific antibody according to the third aspect of the invention are examples of the multivalent.
  • the anti-PD-L2 Nanobody according to the first aspect of the present invention there is provided the anti-PD-L1 Nanobody according to the second aspect of the present invention, or the anti-PD-L1 Nanobody according to the third aspect of the present invention.
  • the bispecific antibody or the use of the immunoconjugate according to the eighth aspect of the present invention, is used for the preparation of medicaments, reagents, detection plates or kits; wherein, the reagents, detection plates or kits are used for : Detection of PD-L1 and/or PD-L2 in the sample; wherein the agent is used to treat or prevent tumors expressing PD-L1 (ie, PD-L1 positive) or tumors expressing PD-L2.
  • the coupling part of the immunoconjugate is an isotope for diagnosis.
  • the reagent is one or more reagents selected from the following group: isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano magnetic particle and imaging agent .
  • the reagent for detecting PD-L1 and/or PD-L2 in the sample is a contrast agent for detecting PD-L1 and/or PD-L2 molecules (in vivo).
  • the detection is in vivo detection or in vitro detection.
  • the detection includes flow cytometry and cellular immunofluorescence detection.
  • the medicament is used to block the interaction between PD-1 and PD-L1, and/or block the interaction between PD-1 and PD-L2.
  • the tumor includes but is not limited to: acute myeloid leukemia, chronic myelogenous leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , Liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumors, bladder tumors.
  • a pharmaceutical composition which contains: (i) the anti-PD-L2 nanobody according to the first aspect of the present invention, and the anti-PD-L1 antibody according to the second aspect of the present invention. Nanobody, or bispecific antibody according to the third aspect of the invention, or immunoconjugate according to the eighth aspect of the invention; and (ii) a pharmaceutically acceptable carrier.
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.
  • the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, levatinib, and pembrolizumab.
  • the drug is used to block the interaction between PD-1 and PD-L1, and/or block the interaction between PD-1 and PD-L2.
  • the pharmaceutical composition is used to block the PD-1/PD-L1 and/or PD-1/PD-L2 signaling pathway.
  • the pharmaceutical composition is used to treat tumors that express PD-L1 protein (ie, PD-L1 positive) and/or express PD-L2 protein (ie, PD-L2 positive).
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare drugs for preventing and treating tumors.
  • an anti-PD-L2 Nanobody according to the first aspect of the present invention, an anti-PD-L1 Nanobody according to the second aspect of the present invention, or an anti-PD-L1 Nanobody according to the third aspect of the present invention includes:
  • the tumor is a tumor that expresses PD-L1 protein (ie, PD-L1 positive) and/or expresses PD-L2 protein (ie, PD-L2 positive).
  • the use is non-diagnostic and non-therapeutic.
  • the antibody is an antibody against PD-L1 and/or PD-L2.
  • a recombinant protein having: (i) the anti-PD-L2 Nanobody as described in the first aspect of the present invention, as described in the second aspect of the present invention The anti-PD-L1 Nanobody, or the bispecific antibody as described in the third aspect of the present invention; and (ii) optionally a tag sequence that assists in expression and/or purification.
  • the tag sequence includes 6His tag, HA tag and Fc tag.
  • the recombinant protein specifically binds to PD-L1 and/or PD-L2.
  • a method for detecting PD-L1 and/or PD-L2 in a sample includes the steps of: (1) combining the sample with the anti-PD-L1 and/or PD-L2 in the first aspect of the present invention.
  • a method for treating diseases comprising administering to a subject in need the anti-PD-L2 Nanobody as described in the first aspect of the present invention, as in the second aspect of the present invention
  • the subject includes mammals, preferably humans.
  • a PD-L1 and/or PD-L2 detection reagent is provided, and the detection reagent comprises the immunoconjugate according to the eighth aspect of the present invention and is detectably acceptable a.
  • the coupling part of the immunoconjugate is an isotope for diagnosis.
  • the detection-acceptable carrier is a non-toxic and inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the following group: isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano magnetic particle and imaging Agent.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (such as liquid, injection, lyophilized powder, tablet, buccal, or mist).
  • kits for detecting PD-L1 and/or PD-L2 the kit containing the immunoconjugate according to the eighth aspect of the present invention or the immunoconjugate according to the eighth aspect of the present invention.
  • the instructions stated that the kit is used to non-invasively detect the expression of PD-L1 and/or PD-L2 of the test subject.
  • the kit is used for the detection of tumors expressing PD-L1 protein (ie, PD-L1 positive) and/or expressing PD-L2 protein (ie, PD-L2 positive).
  • Figure 1 shows the binding activity of purified anti-PD-L2 antibodies to CHO-hPD-L2 cells.
  • Figure 2 shows the blocking effect of the purified anti-PD-L2 antibody on the binding of PD-L2 to PD-1.
  • Figure 3 shows the binding activity of D-Na-96 humanized antibody to CHO-hPD-L2 cells.
  • Figure 4 shows the blocking effect of D-Na-96 humanized antibody on the binding of PD-L2 to PD-1.
  • Figure 5 shows the binding activity of humanized anti-PD-L1 Nanobodies to CHO-hPD-L1 cells.
  • Figure 6 shows the schematic structure of three different forms of anti-PD-L1/PD-L2 bispecific antibodies.
  • Figure 7 shows the binding activity of the anti-PD-L1/PD-L2 bispecific antibody of the present invention to CHO-hPD-L1 cells ( Figure 7A) or CHO-hPD-L2 cells ( Figure 7B).
  • Figure 8 shows the binding of the anti-PD-L1/PD-L2 bispecific antibody of the present invention to PD-L1 and PD-1 ( Figure 8A) and the inhibition of the binding of PD-L2 to PD-1 ( Figure 8B) Effect.
  • Figure 9 shows the blocking effect of the anti-PD-L1/PD-L2 bispecific antibody on the PDL1/PDL2/PD1/luc signaling pathway.
  • an anti-PD-L1/PD-L2 bispecific antibody for the first time, which includes anti-PD-L1 single domain antibody and anti-PD-L2 single domain antibody .
  • the bispecific antibody of the present invention has good binding activity to both PD-L1 and PD-L2 molecules, and can block the interaction between PD-1 and PD-L1 and the interaction between PD-1 and PD-L2. It interacts and can simultaneously block the PD-L1/PD-1 and PD-L2/PD-1 signaling pathways in vitro and activate the expression of downstream reporter genes, thus having good anti-tumor activity.
  • the present invention has been completed on this basis.
  • bispecific antibody of the present invention As used herein, the terms "bispecific antibody of the present invention”, “biantibody of the present invention”, and “anti-PD-L1/PD-L2 bispecific antibody” have the same meaning, and all refer to specific recognition and binding of PD -Bispecific antibodies of L1 and PD-L2.
  • the present invention provides an anti-PD-L1/PD-L2 bispecific antibody, which includes: an anti-PD-L1 single domain antibody and an anti-PD-L2 single domain antibody.
  • the bispecific antibody of the present invention contains a polypeptide having a structure shown in Formula I or Formula II, or a polypeptide having a structure shown in Formula III and Formula IV at the same time,
  • a and B are each independently an anti-PD-L1 single domain antibody or an anti-PD-L2 single domain antibody, and A and B are different antibodies;
  • L1, L2, L3 and L4 are each independently a peptide bond or linker element
  • Fc1 and Fc2 are the Fc segments of antibodies; wherein Fc1 is a human IgG domain (preferably a LALA mutant IgG domain), and Fc2 is a CH1+CL domain;
  • the bispecific antibody has a polypeptide having the structure shown in Formula I, and the polypeptide forms a homodimer i through the disulfide bond between Fc1.
  • the bispecific antibody has a polypeptide having the structure shown in Formula II, and the polypeptide forms a homodimer ii through the disulfide bond between Fc1.
  • the bispecific antibody has the polypeptide sequence of the structure shown in formula III and formula IV, and the polypeptide of the structure shown in formula III and the polypeptide of the structure shown in formula IV pass through Fc2.
  • the inter-disulfide bond action forms a heterodimer a, and the heterodimer ii forms a homodimer iii through the disulfide bond action between Fc1.
  • single domain antibody As used herein, the terms “single domain antibody”, “Nanobody VHH”, and “Nanobody” have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a Nanobody consisting of only one heavy chain variable region ( VHH), it is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody with naturally missing light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a -folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partially folded structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%. , 97%, 98% or 99%.
  • human framework region is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .
  • affinity is theoretically defined by the balanced association between the intact antibody and the antigen.
  • the affinity of the double antibody of the present invention can be evaluated or determined by the KD value (dissociation constant) (or other measurement methods), such as Bio-layer Interferometry (BLI), which is measured and determined by the FortebioRed96 instrument.
  • KD value dissociation constant
  • BLI Bio-layer Interferometry
  • linker refers to the insertion of an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold to exchange one or more amino acid residues of the immunoglobulin with dual variable regions. base.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form ⁇ conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the PD-L1/PD-L2 bispecific antibody or fragments thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • the terms "antibody of the present invention”, “protein of the present invention”, or “polypeptide of the present invention” are used interchangeably, and all refer to a polypeptide that specifically binds to PD-L1 and/or PD-L2 protein, for example, has a heavy
  • the protein or polypeptide of the variable region of the chain may or may not contain the starting methionine.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention.
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homology.
  • variable regions which are divided into 4 framework regions (FR), 4 FR amino acids
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • 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 antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified here. sex.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the antibody of the present invention refers to a double antibody with PD-L1 and/or PD-L2 protein binding activity.
  • the term also includes variant forms of polypeptides that have the same functions as the antibodies of the present invention and include the same CDR regions. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the present invention also provides other polypeptides, such as fusion proteins containing single domain antibodies or fragments thereof.
  • the present invention also includes fragments of single domain antibodies of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • 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 Preferred substitution Ala(
  • the present invention also provides polynucleotide molecules encoding the above-mentioned 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 synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and 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 that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • One feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutics ( For example, cisplatin) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-L1 and/or PD-L2 protein molecules, and thus can be used to treat tumors.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned single domain antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the antibody carries a detectable label. More preferably, the label is selected from the following group: isotope, colloidal gold label, colored label or fluorescent label.
  • Colloidal gold labeling can be performed by methods known to those skilled in the art.
  • the PD-L1/PD-L2 bispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
  • the present invention also relates to a method for detecting PD-L1 and/or PD-L2 protein.
  • the method steps are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of PD-L1 and/or PD-L2 protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a sample containing cells in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffers and the like.
  • the present invention also provides a detection kit for detecting the level of PD-L1 and/or PD-L2.
  • the kit includes an antibody that recognizes the PD-L1 and/or PD-L2 protein, used to dissolve the lysis medium of the sample, and detect Required general reagents and buffers, such as various buffers, detection labels, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the single domain antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves the diagnosis and treatment of diseases related to PD-L1 and/or PD-L2, basic medical research, biological research, etc. Multiple areas.
  • a preferred application is for clinical diagnosis and targeted therapy for PD-L1 and/or PD-L2, such as tumor treatment.
  • Nanobody of the present invention is highly specific to human PD-L1 protein with the correct spatial structure.
  • Nanobody of the present invention is highly specific to human PD-L2 protein with correct spatial structure.
  • the Nanobody of the present invention has strong affinity.
  • the present invention can simultaneously block the interaction of PD-L1/PD-1 and PD-L2/PD-1, relieve immunosuppression, activate the body's immune system to kill tumors.
  • IgG2 and IgG3 sequences were amplified from cDNA:
  • the PCR products were subjected to agarose gel electrophoresis, and the fragments at 750 bp were recovered from the gel for the second round of VHH sequence amplification.
  • the primers for the second round of PCR amplification are as follows:
  • a third round of PCR was performed, adding homology arms to the VHH gene.
  • the primers for the third round of PCR amplification are as follows:
  • the target fragment was recovered using PCR purification kit (purchased from QIAGEN).
  • the linearized yeast display vector and the third round of PCR products were mixed and electrotransformed into Saccharomyces cerevisiae (purchased from ATCC).
  • Saccharomyces cerevisiae purchased from ATCC.
  • the anti-PD-L2 nanobody library from two animals was constructed and the storage capacity was determined.
  • the storage capacity was 4.47. ⁇ 10 7 and 4.14 ⁇ 10 7 .
  • MACS enriches yeasts that specifically bind to PD-L2
  • Biotin-labeled PD-L2 protein (final concentration 100mM), incubate at room temperature for 30min, collect yeast cells by centrifugation, and wash the yeast 3 times with 50ml washing solution. Resuspend the yeast cells with 5ml washing solution, and add 200 ⁇ l SA magnetic beads (purchased from Miltenyi), and incubate the cells upside down for 10min. Wash the yeast and magnetic beads mixture 3 times with the cleaning solution, and add the mixture to the LS purification column (purchased from Miltenyi). Place the LS purification column on the magnetic stand, and wash the non-specifically bound yeast cells with the washing solution. Take the purification column out of the magnetic stand and add the washing solution to elute the yeast. The eluted yeast is centrifuged and transferred to 200ml SD-CAA amplification medium for amplification.
  • Use SD-CAA induction medium (1L SD-CAA induction medium contains 6.7g YNB, 5g tyrosine, 13.62g Na 2 HPO 4 ⁇ 12H 2 O, 7.44g NaH 2 PO 4 and 2% galactose, 2% Raffinose and 0.1% glucose) resuspend yeast cells at an initial concentration of 0.5 OD 600 /ml, and induce overnight.
  • the yeast solution with high binding ability to PD-L2 antigen obtained by MACS and FACS enrichment was cultured in SD-CAA amplification medium at 30°C and 225rpm overnight.
  • yeast plasmid extraction kit purchased from Tiangen
  • the plasmid was transformed into Top10 competent cells (purchased from Tiangen) by electroporation, coated with ampicillin resistant plates, and cultured at 37°C overnight. Pick a single clone for sequencing to obtain the VHH gene sequence.
  • the VHH gene sequence was connected to the Fc segment of human IgG1 (LALA mutation), and the homologous recombinase was used to construct the pCDNA3.1 vector (purchased from Vazyme) and EcoR I/Not I double digestion linearized pCDNA3.1 vector.
  • the homologous recombination product was transformed into Top10 competent cells, coated with ampicillin resistant plates, incubated overnight at 37°C, single clones were picked for sequencing, and plasmids were extracted.
  • ExpiCHO TM expression system kit purchased from Thermo
  • the extracted plasmid was transferred into Expi-CHO cells.
  • the transfection method was in accordance with the commercial instructions. After 5 days of cell culture, the supernatant was collected using protein A magnetic beads (purchased from GenScript). ) Purification of the target protein by sorting method.
  • the magnetic beads were resuspended (1-4 times the volume of the magnetic beads) with an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, incubated at room temperature for 1 hour, and gently shaken during the period.
  • binding buffer PBS+0.1% Tween 20, pH 7.4
  • the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer.
  • elution buffer 0.1M sodium citrate, pH 3.2
  • elution buffer 0.1M sodium citrate, pH 3.2
  • the pCHO1.0 vector (purchased from Invitrogen) cloned human PD-L2cDNA (purchased from Sino Biological) was transfected to produce CHO cells (CHO-hPD-L2 cells) overexpressing human PD-L2. Adjust the cell density of the expanded CHO-hPD-L2 cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for use. Dilute the purified PD-L2 antibody with PBS, start 3-fold dilution at 1000 nM, a total of 12 points, add 100 ⁇ l/well of the above diluted sample to the above 96-well flow plate with cells, incubate at 4°C for 30 minutes, and wash with PBS twice.
  • ForteBio affinity determination is performed in accordance with existing methods (Estep, P et al., determination of antibody-antigen affinity and epitope binding based on a high-throughput method. MAbs, 2013.5(2): p.270-8).
  • the sensor is equilibrated offline in the analysis buffer for 30 minutes, and then tested online for 60 seconds to establish a baseline, and the purified antibody obtained as described above is loaded online onto the AHQ sensor. Then put the sensor in 100nM PD-L2 antigen for 5min, then transfer the sensor to PBS to dissociate for 5min.
  • a 1:1 combination model was used for kinetic analysis.
  • the pCHO1.0 vector purchased from Invitrogen
  • cloned human PD-1 cDNA purchased from Sino Biological
  • APC goat anti-mouse IgG (minimal x reactive) antibody purchased from Biolegend
  • APC goat anti-mouse IgG (minimal x reactive) antibody purchased from Biolegend
  • PBS fetal bovine serum
  • 100 ⁇ l/well was added to PBS to resuspend the cells, detected on a CytoFlex (Bechman) flow cytometer, and calculated the corresponding MFI.
  • D-NA-96 and D-Ye-29 antibodies were humanized.
  • the humanization method adopts the VHH humanized universal framework transplantation method, and at the same time according to the literature (Vincke, C., et al., humanized camel single domain antibodies and the general strategy for identifying universal humanized nanobody scaffolds. J Biol Chem 284 (5):3273-3284) The method reported to complete the mutation of some amino acids of antibody framework 2 (framework2).
  • IMGT http://www.imgt.org
  • IMGT http://www.imgt.org
  • the protein construction and expression purification methods were the same as in Example 1.3, and the purity of the obtained protein was detected by HPLC.
  • the HPLC method is as follows, mobile phase: 150 mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m).
  • This experiment detects the binding activity of the purified humanized D-Na-96 sample with CHO-hPD-L2 cells.
  • the experimental method is the same as that in Example 1.4.
  • the experimental results are shown in Figure 3.
  • the humanized D-Na-96 sample is CHO-hPD-L2 cells have good binding activity, and the level is equivalent to D-Na-96.
  • This experiment detects the binding affinity of the purified humanized D-Na-96/D-Ye-29 sample to human PD-L2.
  • the experimental method is the same as that in Example 1.5.
  • the experimental results are shown in Table 7. D-Na-96/ D-Ye-29 humanized samples have good binding activity with human PD-L2 protein.
  • Nano-library construction methods are the same as in Example 1.1.
  • Example 1.2 Nanobody sequences that can specifically bind to human PD-L1 were screened out, and the specific screening method was the same as that in Example 1.2.
  • the VHH gene sequence and the Fc segment of human IgG1 (LALA mutation) were connected and constructed into the eukaryotic expression vector pCDNA3.1.
  • the ExpiCHO expression system and magnetic bead affinity purification system were used to prepare high-purity heavy chain antibody proteins.
  • the construction, expression and purification methods of heavy chain antibody pairs are the same as in Example 1.3.
  • K-Yr-13&14-02 antibodies were humanized.
  • the humanization method is the same as in Example 1.7.
  • Example 1.3 The protein construction, expression and purification and HPLC purity detection methods are the same as in Example 1.3. The results are shown in Table 10. After one-step purification, a humanized anti-PD-L1 heavy chain antibody protein with higher purity was obtained.
  • This experiment detects the binding activity of the purified humanized sample HZ-K-Yr-13&14-02-3 to CHO-hPD-L1 cells.
  • the experimental method is the same as that in Example 1.4.
  • the experimental results are shown in Figure 5.
  • HZ-K- Yr-13&14-02-3 has good binding activity to CHO-hPD-L1 cells, and the level is equivalent to K-Yr-13&14-02 and the control antibody ATE.
  • This experiment detects the binding affinity of the purified HZ-K-Yr-13&14-02-3 to human PD-L1.
  • the experimental method is the same as that in Example 1.5.
  • the experimental results are shown in Table 11. HZ-K-Yr-13&14-02- 3 It has good binding activity with human PD-L2 protein.
  • Bi-201 contains a peptide chain with the amino acid sequence shown in SEQ ID NO: 23, which includes the anti-PD-L1 Nanobody HZ-K-Yr-13&14-02-3, the C-terminal of the Nanobody amino acid sequence and The domains of human IgG1 (LALA mutant) are directly connected.
  • the anti-PD-L2 Nanobody HZ-D-NA-96-1 is connected to the C-terminus of Fc through a flexible peptide chain (GGGGSGGGGSGGGGSGGGGSG) (SEQ ID NO: 27).
  • Bi-202 contains a peptide chain with the amino acid sequence shown in SEQ ID NO: 24, the C-terminus of the anti-PD-L1 Nanobody HZ-K-13&14-02-03 amino acid sequence and the anti-PD-L2 Nanobody HZ- D-NA-96-1 is connected by a flexible peptide chain (GGGGSGGGGSGGGGSGGGGSG) (SEQ ID NO: 27). The C-terminus of HZ-D-NA-96-1 is directly connected to the human IgG1 (LALA mutant) domain.
  • Bi-203-204 contains two peptide chains.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 25, the C-terminus of the anti-PD-L1 Nanobody HZ-K-Yr-13&14-02-3 amino acid sequence and the amino acid sequence of SEQ ID NO: 29 derived from human IgG1 Shows that the CH1 amino acid sequence is directly connected; the human IgG1 (LALA mutant) Fc (SEQ ID NO:) domain is directly connected to the C-terminus of the CH1 region, thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 26, which includes the anti-PD-L2 Nanobody HZ-D-NA-96-01 amino acid sequence SEQ ID NO: 16, and the C-terminal of the Nanobody amino acid sequence is directly The human kappa light chain constant region (CL) amino acid sequence SEQ ID NO: 30 is connected to obtain peptide chain #2.
  • the nucleotide sequences encoding the anti-PD-L1/PD-L2 bispecific antibodies Bi-201, Bi-202, and Bi-203-204 constructed in Example 3.1 were all passed through the multiple cloning site Connect into the commercially available eukaryotic expression vector pCDNA3.1(+).
  • the ExpiCHO expression system and magnetic bead affinity purification system were used to prepare high-purity heavy chain antibody proteins.
  • the protein construction, expression and purification and HPLC purity detection methods are the same as in Example 1.3. The results are shown in Table 12. After one-step purification, a bispecific antibody protein with higher purity was obtained.
  • the ForteBio instrument was used to detect the binding activity of the obtained anti-PD-L1/PD-L2 bispecific antibody and human PD-L1 protein or human PD-L2, and the detection method was the same as in Example 1.5.
  • the test results are shown in Table 13 and Table 14.
  • the three candidate molecules obtained in this study have good binding activity with human PD-L1 and human PD-L2 proteins.
  • This experiment detects the binding activity of the purified anti-PD-L1/PD-L2 bispecific antibody to CHO-hPD-L1 cells or CHO-hPD-L2.
  • the experimental method is the same as that in Example 1.4.
  • the experimental results are shown in Figures 7A and 7B.
  • Bi-201, Bi-202, Bi-203-204 have good binding activity with CHO-hPD-L1 cells and CHO-hPD-L2 cells.
  • Anti-PD-L1/PD-L2 bispecific antibody blocks the binding of PD-L2/PD-L1 to PD-1
  • PD-L1 and PD-L2 can be co-expressed on tumor cells or immune cells.
  • This example uses CHO cells that co-express human PD-L1 and human PD-L2 (CHO-K1-PD-L1/PD-L2) and The method of co-incubation of Jurkat cells (Jurkat-PD-1-NFAT) overexpressing human PD-1 and containing the NFAT-luciferase reporter gene detected the effects of purified antibodies Bi-201, Bi-202, and Bi-203-204 on PD -Simultaneous blocking of L1/PD-1 pathway and PD-L2/PD-1 pathway, the specific method is as follows.
  • CHO-K1-PD-L1/PD-L2 cells Adjust the density of CHO-K1-PD-L1/PD-L2 cells to 5 ⁇ 10 5 cells/ml, inoculate 100 ⁇ l/well on a 96-well cell culture white bottom plate, and place them in a 37°C, 5% CO 2 incubator for overnight culture. After the purified antibody and the control antibody 1640 complete medium were diluted stepwise, they were used for later use. Adjust the cell density of Jurkat-PD-1-NFAT cells to 2.5 ⁇ 10 5 cells/ml with 1640 complete medium for use.
  • the experimental results are shown in Figure 9.
  • the anti-PD-L1/PD-L2 bispecific antibody of the present invention can simultaneously block the PD-L1/PD-1 and PD-L2/PD-1 signal pathways in vitro, and activate the expression of downstream reporter genes.
  • Neither anti-PD-L1 nor anti-PD-L2 monoclonal antibodies can completely block this pathway and activate the expression of downstream reporter genes.

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Abstract

提供了PD-L1纳米抗体、PD-L2纳米抗体和同时具有PD-L1纳米抗体和PD-L2纳米抗体的双特异性抗体，该双特异性抗体可以同时阻断PD-1/PD-L1及PD-1/PD-L2通路。本发明的双特异性抗体可以使T细胞恢复活性，增强免疫应答，更有效地提高对肿瘤发生和发展的抑制效果。

Description

一种抗PD-L1和PD-L2抗体及其衍生物和用途 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种抗PD-L1和PD-L2抗体及其衍生物和用途。
背景技术
程序性死亡因子1(programmed death-1，PD-1)又称CD279，是CD28家族成员，其胞质区含有2个酪氨酸残基，靠近N端的1个位于免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif，ITIM)中，靠近C端的1个位于免疫受体酪氨酸转化基序(immunoreceptor tyrosine-based switch motif，ITSM)中。PD-1主要表达在活化的T淋巴细胞、B淋巴细胞和巨噬细胞表面。在正常情况下，PD-1能够抑制T淋巴细胞的功能，促进Treg的功能，从而抑制自身免疫应答，防止自身免疫性疾病的发生。
程序性死亡因子1配体1(programmed death 1 ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。
与其他B7家族分子不同的是，PD-L1具有负向调节免疫应答的作用。研究发现，PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等，除淋巴细胞外，PD-L1也表达于其他多种组织如胸腺、心脏、胎盘等的内皮细胞，以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性，并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。
但在肿瘤的发生中，肿瘤细胞表达的PD-L1与PD-1结合后却能通过对淋巴细胞的抑制性作用促进肿瘤的免疫逃逸。PD-L1与PD-1的结合可导致多种生物学变化，引起免疫调控，如能够抑制淋巴细胞的增殖和活化、抑制CD4+T细胞向Th1和Th17细胞分化、抑制炎性细胞因子的释放等。
程序性死亡因子1配体2(programmed death 1 ligand 1，PD-L2)又称CD273或B7-DC，是PD-1的另一个重要的配体。PD-1和PD-L2的相互作用会抑制CD4+ T细胞的活化及增殖、减少细胞因子的释放，增加肿瘤的免疫逃逸。同时阻断PD-1和PD-L1及PD-L2的相互作用可以有效解放PD-1通路活化对免疫***的抑制，激活机体自身免疫***，杀灭肿瘤。
然而，截止目前，市场上尚未有同时靶向PD-L1/PD-L2的双特异性单域抗体公开，而单域抗体作为新一代抗体诊断及治疗中的新兴力量，具有稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等特点，在免疫实验、诊断与治疗中，发挥着超乎想象的巨大功能。
因此，本领域迫切需要一种能够同时靶向PD-L1和PD-L2的双特异性单域抗体。
发明内容
本发明的目的就是提供一种能够同时靶向PD-L1和PD-L2的双特异性抗体。
在本发明的第一方面，提供了一种抗PD-L2纳米抗体，所述PD-L2纳米抗体的VHH链的各互补决定区CDR由以下组成：
氨基酸序列如SEQ ID NO:57所示的CDR1；氨基酸序列如SEQ ID NO:58所示的CDR2；和氨基酸序列如SEQ ID NO:59所示的CDR3；或
氨基酸序列如SEQ ID NO:60所示的CDR1；氨基酸序列如SEQ ID NO:61所示的CDR2；和氨基酸序列如SEQ ID NO:62所示的CDR3；
或者，所述抗PD-L2纳米抗体的VHH链的氨基酸序列如SEQ ID NO:1、2、4、5、6、7、8、9、10、11、13、14、17或18所示。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的抗PD-L2纳米抗体的VHH链的氨基酸序列如SEQ ID NO:3(即D-Na-96)、16(即HZ-D-Na-96-1)、12(即D-Ye-29)或15(即HZ-D-Ye-29-3)所示。
在另一优选例中，所述的抗PD-L2纳米抗体是经人源化的，并且所述抗PD-L2纳米抗体的VHH链的氨基酸序列如16(即HZ-D-Na-96-1)或15(即HZ-D-Ye-29-3) 所示。
在另一优选例中，所述的PD-L2纳米抗体可以阻断PD-1和PD-L2的相互作用。
在本发明的第二方面，提供了一种抗PD-L1纳米抗体，所述PD-L1纳米抗体的VHH链的各互补决定区CDR由以下组成：
氨基酸序列如SEQ ID NO:63所示的CDR1；氨基酸序列如SEQ ID NO:64所示的CDR2；和氨基酸序列如SEQ ID NO:65所示的CDR3。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的抗PD-L1纳米抗体的VHH链的氨基酸序列如SEQ ID NO:19(即K-Yr-13&14-02)、20(即K-Yr-13&14-09)、21(即K-Yr-13&14-16)或22(即HZ-K-Yr-13&14-02-3)所示。
在另一优选例中，所述的抗PD-L1纳米抗体是经人源化的，并且所述抗PD-L1纳米抗体的VHH链的氨基酸序列如SEQ ID NO:22(即HZ-K-Yr-13&14-02-3)所示。
在另一优选例中，所述的PD-L1纳米抗体可以阻断PD-1和PD-L1的相互作用。
在本发明的第三方面，提供了一种双特异性抗体，所述双特异性抗体包括：如本发明第二方面所述的抗PD-L1纳米抗体和如本发明第一方面所述的抗PD-L2纳米抗体。
在另一优选例中，所述的双特异性抗体包括1-3个抗PD-L1纳米抗体，较佳地，包括1或2个抗PD-L1纳米抗体。
在另一优选例中，所述的双特异性抗体包括1-3个抗PD-L2纳米抗体，较佳地，包括1或2个抗PD-L2纳米抗体。
在另一优选例中，所述的双特异性抗体还包含Fc段。
在另一优选例中，所述双特异性抗体的Fc段选自下组：人IgG结构域、CH1+CL1结构域，或其组合。
在另一优选例中，所述人IgG结构域是经过改造的突变型IgG结构域，优选地为LALA突变型的IgG结构域。
在另一优选例中，所述的双特异性抗体含有如式I或式II所示结构的多肽，或同时含有如式III和式IV所示结构的多肽，
A-L1-Fc1-L2-B      (式I)
A-L3-B-L4-Fc1      (式II)
A-L5-Fc2-L6-Fc1      (式III)
B-L7-Fc2         (式IV)
其中，
A和B各自独立地为如本发明第二方面所述的抗PD-L1纳米抗体或如本发明第一方面所述的抗PD-L2纳米抗体，且A和B是不同的抗体；
L1、L2、L3和L4各自独立地为肽键或接头元件；
Fc1、Fc2均为抗体的Fc段；其中Fc1为人IgG结构域(优选地为LALA突变型IgG结构域)，并且Fc2为CH1+CL结构域；
“-”为肽键。
在另一优选例中，所述的双特异性抗体具有如式III和式IV所示结构的多肽序列，并且所述式III所示结构的多肽和式IV所示结构的多肽之间通过二硫键形成异源二聚体a。
在另一优选例中，所述双特异性抗体具有式I所示结构的多肽，且所述多肽通过Fc1之间的二硫键作用形成同源二聚体i。
在另一优选例中，所述双特异性抗体具有式II所示结构的多肽，且所述多肽通过Fc1之间的二硫键作用形成同源二聚体ii。
在另一优选例中，所述的双特异性抗体具有如式III和式IV所示结构的多肽序列，并且所述式III所示结构的多肽和式IV所示结构的多肽之间通过Fc2之间的二硫键作用形成异源二聚体a，并且所述异源二聚体ii通过Fc1之间的二硫键作用形成同源二聚体iii。
在另一优选例中，所述的PD-L1纳米抗体的氨基酸序列如SEQ ID NO：19、20、21或22所示，优选地为SEQ ID NO:22。。
在另一优选例中，所述的双特异性抗体还包括其他抗PD-L2纳米抗体的VHH链，所述的其他PD-L2纳米抗体的氨基酸序列如SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18所示。
在另一优选例中，所述的抗PD-L2纳米抗体的VHH链的氨基酸序列如SEQ ID NO:3、16、12或15所示，优选地为SEQ ID NO:16或15。
在另一优选例中，所述的接头元件的序列为(4GS)n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝4。
在另一优选例中，所述接头元件的序列如SEQ ID NO：27所示，或与SEQ ID NO：27所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述LALA突变型的人IgG结构域的氨基酸序列如SEQ ID NO：28所示，或与SEQ ID NO：28所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述CH1结构域的氨基酸序列如SEQ ID NO：29所示，或与SEQ ID NO：29所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述CL结构域的氨基酸序列如SEQ ID NO：30所示，或与SEQ ID NO：30所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述双特异性抗体的氨基酸序列如SEQ ID NO：23或24所示。
在另一优选例中，所述双特异性抗体同时含有如式III和式IV所示结构的多肽，其中所述的如式III所述的多肽的氨基酸序列如SEQ ID NO：25所示，所述的如式IV所述的多肽的氨基酸序列如SEQ ID NO：26所示。
在本发明的第四方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体。
在另一优选例中，所述多核苷酸的序列如SEQ ID NO:31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47或48所示。
在另一优选例中，所述多核苷酸的序列如SEQ ID NO:33(即D-Na-96)、46(即HZ-D-Na-96-1)、42(即D-Ye-29)或45(即HZ-D-Ye-29)所示；优选地为SEQ ID NO:46或45。
在另一优选例中，所述多核苷酸的序列如SEQ ID NO:49(即K-Yr-13&14-02)、50(即K-Yr-13&14-09)、51(即K-Yr-13&14-16)或52(即HZ-K-Yr-13&14-02-3)所示；优选地为SEQ ID NO:52。
在另一优选例中，所述多核苷酸的序列如SEQ ID NO:53或54所示。
在另一优选例中，所述多核苷酸包括序列如SEQ ID NO:55所示的第一多核苷酸和序列如SEQ ID NO:56所示的第二多核苷酸。
在本发明的第五方面，提供了一种载体，所述载体含有如本发明第四方面所 述的多核苷酸。
在另一优选例中，所述的载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移***、或其组合；优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
在本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第五方面所述的载体，或其基因组中整合有如本发明第四方面所述的多核苷酸；
或者，所述的宿主细胞表达如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
在本发明的第七方面，提供了一种产生抗PD-L1纳米抗体、抗PD-L2纳米抗体，或双特异性抗体的方法，包括步骤：
(a)在合适的条件下，培养如本发明第六方面所述的宿主细胞，从而获得含所述抗PD-L1纳米抗体、抗PD-L2纳米抗体，或双特异性抗体的培养物；和
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的抗PD-L1纳米抗体、抗PD-L2纳米抗体，或双特异性抗体。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
在本发明的第八方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：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、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：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-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(如DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(如顺铂)。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体。
在本发明的第九方面，提供了如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体，或如本发明第八方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1和/或PD-L2；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤或是表达PD-L2的肿瘤。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述检测样品中PD-L1和/或PD-L2的试剂为(体内)检测PD-L1和/或PD-L2分子的造影剂。
在另一优选例中，所述的检测为体内检测或体外检测。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
在另一优选例中，所述的药剂用于阻断PD-1和PD-L1的相互作用，和/或阻断PD-1和PD-L2的相互作用。
在另一优选例中，所述的肿瘤包括但不限于：急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、***癌、***、淋巴癌、肾上腺肿瘤、***。
在本发明的第十方面，提供了一种药物组合物，含有：(i)如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体，或如本发明第八方面所述的免疫偶联物；以及(ii)药学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。
在另一优选例中，所述的药物组合物中还含有***的其他药物，如细胞毒性药物。
在另一优选例中，所述的***的其他药物包括紫杉醇、多柔比星、环磷酰胺、阿西替尼、乐伐替尼、派姆单抗。
在另一优选例中，所述的药物用于阻断PD-1和PD-L1的相互作用，和/或阻断PD-1和PD-L2的相互作用。
在另一优选例中，所述的药物组合物用于阻断PD-1/PD-L1和/或PD-1/PD-L2信号通路。
在另一优选例中，所述的药物组合物用于治疗表达PD-L1蛋白(即PD-L1阳性)和/或表达PD-L2蛋白(即PD-L2阳性)的肿瘤。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备防治肿瘤的药物。
在本发明的第十一方面，提供了如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体的一种或多种选自下组的用途，包括：
(i)用于检测人PD-L1分子和/或PD-L2分子；(ii)用于流式检测；(iii)用于细胞免疫荧光检测；(iv)用于***；(v)用于肿瘤诊断；(vi)用于阻断PD-1和PD-L1的相互作用；和/或(vii)用于阻断PD-L2和PD-1的相互作用。
在另一优选例中，所述的肿瘤为表达PD-L1蛋白(即PD-L1阳性)和/或表达PD-L2蛋白(即PD-L2阳性)的肿瘤。
在另一优选例中，所述用途为非诊断的和非治疗的。
在另一优选例中，所述的抗体为抗PD-L1和/或PD-L2的抗体。
在本发明的第十二方面，提供了一种重组蛋白，所述的重组蛋白具有：(i)如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体；以及(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签、HA标签和Fc标签。
在另一优选例中，所述的重组蛋白特异性结合于PD-L1和/或PD-L2。
在本发明的第十三方面，提供了一种检测样品中PD-L1和/或PD-L2的方法，所述方法包括步骤：(1)将样品与如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体接触；(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PD-L1和/或PD-L2。
在本发明的第十四方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用如本发明第一方面所述的抗PD-L2纳米抗体、如本发明第二方面所述的抗PD-L1纳米抗体，或如本发明第三方面所述的双特异性抗体、如本发明第八方面所述的免疫偶联物，或如本发明第十方面所述的药物组合物。
在另一优选例中，所述的对象包括哺乳动物，优选地是人。
在本发明的第十五方面，提供了一种PD-L1和/或PD-L2检测试剂，所述的检测试剂包含如本发明第八方面所述的免疫偶联物和检测学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述的检测试剂用于体内检测。
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂，针剂，冻干粉，片剂，含服剂，吸雾剂)。
在本发明的第十六方面，提供了一种检测PD-L1和/或PD-L2的试剂盒，所 述试剂盒含有如本发明第八方面所述的免疫偶联物或如本发明第十五方面所述的检测试剂，以及说明书。
在另一优选例中，所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的PD-L1和/或PD-L2表达。
在另一优选例中，所述的试剂盒用于表达PD-L1蛋白(即PD-L1阳性)和/或表达PD-L2蛋白(即PD-L2阳性)的肿瘤的检测。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了纯化的抗PD-L2抗体与CHO-hPD-L2细胞的结合活性。
图2显示了纯化的抗PD-L2抗体对PD-L2与PD-1结合的阻断效果。
图3显示了D-Na-96人源化抗体与CHO-hPD-L2细胞的结合活性。
图4显示了D-Na-96人源化抗体对PD-L2与PD-1结合的阻断效果。
图5显示了人源化抗PD-L1纳米抗体与CHO-hPD-L1细胞的结合活性。
图6显示了3种不同形式的抗PD-L1/PD-L2双特异性抗体的结构示意图。
图7显示了本发明的抗PD-L1/PD-L2双特异性抗体与CHO-hPD-L1细胞(图7A)或CHO-hPD-L2细胞(图7B)的结合活性。
图8显示了本发明的抗PD-L1/PD-L2双特异性抗体对PD-L1与PD-1的结合(图8A)以及对PD-L2与PD-1的结合(图8B)的阻断效果。
图9显示了抗PD-L1/PD-L2双特异性抗体对PDL1/PDL2/PD1/luc信号通路的阻断效果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种抗PD-L1/PD-L2双特异性抗体，其包含抗PD-L1单域抗体和抗PD-L2单域抗体。实验表明，本发明的双特异性抗体对PD-L1和PD-L2分子均具有较好的结合活性，同时能够阻断PD-1与PD-L1的相互作用以及PD-1与PD-L2的相互作用，并且能够在体外同时阻断PD-L1/PD-1和PD-L2/PD-1信号通路，激活下游报告 基因的表达，因而具有良好的抗肿瘤活性。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
双特异性抗体
如本文所用，术语“本发明的双特异性抗体”、“本发明的双抗”、“抗PD-L1/PD-L2双特异性抗体”具有相同的含义，均指特异性识别和结合PD-L1和PD-L2的双特异性抗体。
本发明提供了一种抗PD-L1/PD-L2双特异性抗体，其包括：抗PD-L1单域抗体和抗PD-L2单域抗体。
优选地，本发明的双特异性抗体含有如式I或式II所示结构的多肽，或同时含有如式III和式IV所示结构的多肽，
A-L1-Fc1-L2-B      (式I)
A-L3-B-L4-Fc1      (式II)
A-L5-Fc2-L6-Fc1      (式III)
B-L7-Fc2         (式IV)
其中，
A和B各自独立地为抗PD-L1单域抗体或抗PD-L2单域抗体，且A和B是不同的抗体；
L1、L2、L3和L4各自独立地为肽键或接头元件；
Fc1、Fc2均为抗体的Fc段；其中Fc1为人IgG结构域(优选地为LALA突变型IgG结构域)，并且Fc2为CH1+CL结构域；
“-”为肽键。
在一个实施方式中，所述双特异性抗体具有式I所示结构的多肽，且所述多肽通过Fc1之间的二硫键作用形成同源二聚体i。
在一个实施方式中，所述双特异性抗体具有式II所示结构的多肽，且所述多肽通过Fc1之间的二硫键作用形成同源二聚体ii。
在一个实施方式中，所述的双特异性抗体具有如式III和式IV所示结构的多 肽序列，并且所述式III所示结构的多肽和式IV所示结构的多肽之间通过Fc2之间的二硫键作用形成异源二聚体a，并且所述异源二聚体ii通过Fc1之间的二硫键作用形成同源二聚体iii。
如本文所用，术语“单域抗体”、“纳米抗体VHH”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“框架区”(FR)指***CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明双抗的亲和力可以通过KD值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用FortebioRed96仪器测量确定。
如本文所用，术语“接头”是指***免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的PD-L1/PD-L2双特异性抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1和/或PD-L2蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结 合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1和/或PD-L2蛋白结合活性的双抗。该术语还包括具有与本发明抗体相同功能的、包含相同CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳 地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
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
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以 是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如 果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1和/或PD-L2蛋白分子，因而 可用于***。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的抗体
在本发明的一个优选例中，所述抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，PD-L1/PD-L2双特异性抗体可以用胶体金标记，得到胶体金标记的抗体。
检测方法
本发明还涉及检测PD-L1和/或PD-L2蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1和/或PD-L2蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在 本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-L1和/或PD-L2水平的检测试剂盒，该试剂盒包括识别PD-L1和/或PD-L2蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PD-L1和/或PD-L2相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1和/或PD-L2的临床诊断和靶向治疗，如肿瘤治疗。
本发明的主要优点包括：
(1)本发明纳米抗体高特异性针对人的具有正确空间结构的PD-L1蛋白。
(2)本发明纳米抗体高特异性针对人的具有正确空间结构的PD-L2蛋白。
(3)本发明纳米抗体的亲和力强。
(4)本发明纳米抗体的生产简便。
(5)本发明可以同时阻断PD-L1/PD-1及PD-L2/PD-1的相互作用，解除免疫抑制，激活机体免疫***杀灭肿瘤。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
表B本发明的序列总结
Figure PCTCN2021084196-appb-000001
Figure PCTCN2021084196-appb-000002
Figure PCTCN2021084196-appb-000003
实施例1：抗人PD-L2纳米抗体
1.1纳米抗体文库的构建
动物免疫
将l mg人PD-L2抗原(购自AcroBiosystems)与弗氏佐剂等体积混合，免疫2只羊驼(Llama)，每周一次，共免疫4次，剌激B细胞表达抗原特异性的纳米抗体。4次免疫结束后，提取50ml羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼总cDNA。
纳米抗体基因扩增
第一轮PCR，从cDNA中扩增出IgG2、IgG3序列：
表1.第一轮PCR引物
名称 序列(5’到3’) SEQ ID NO:
上游引物 GTCCTGGCTGCTCTTCTACAAGG 66
下游引物 GGTACGTGCTGTTGAACTGTTCC 67
PCR产物进行琼脂糖凝胶电泳，切胶回收750bp处的片段用于第二轮VHH序列扩增。第二轮PCR扩增引物如下：
表2.第二轮PCR引物
名称 序列(5’到3’) SEQ ID NO:
上游引物 CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT 68
下游引物 CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG 69
以第二轮PCR产物为模板，进行第三轮PCR，为VHH基因加上同源臂。第三轮 PCR扩增引物如下：
表3.第三轮PCR引物
Figure PCTCN2021084196-appb-000004
利用PCR纯化试剂盒(购自QIAGEN)回收目的片段。
文库构建
将线性化的酵母展示载体和第三轮的PCR产物混合后电转化入酿酒酵母(购自ATCC)中，构建来自两个动物的抗PD-L2纳米抗体文库并测定库容，库容大小分别为4.47×10 7和4.14×10 7。
1.2 PD-L2纳米抗体的筛选
人PD-L2蛋白的生物素化标记
取适量体积的双蒸水溶解人PD-L2蛋白(购自AcroBiosystems)，按照生物素标记试剂盒(购自Thermo)产品说明书，将生物素溶解后与蛋白溶液混合，于4℃孵育2小时。用脱盐柱(购自Thermo)去除多余的生物素，脱盐柱预处理及样品收集操作均参考产品说明书步骤进行。
MACS富集能与PD-L2特异性结合的酵母
将实施例1.2中构建的VHH文库接种于SD-CAA扩增培养基(1L SD-CAA扩增培养基中含6.7g YNB、5g酪氨基酸、13.62g Na 2HPO 4·12H 2O、7.44g NaH 2PO 4和2％葡萄糖)中，接种的酵母细胞数>10×文库容量(起始扩增浓度＝0.5OD 600/ml)，30℃，225rpm培养过夜。取10×库容量的酵母细胞，3000rpm×5min离心(以下离心操作均同此)去除培养基，用SD-CAA诱导培养基重悬酵母细胞，调整起始浓度为0.5OD 600/ml，诱导过夜。测定诱导后的文库浓度，取10×库容量的酵母细胞，离心去除培养基。用50ml清洗液(PBS+0.5％BSA+2mM EDTA)重悬酵母细胞，离心去除上清。用10ml清洗液重悬酵母细胞。
加入生物素标记的PD-L2蛋白(终浓度100mM)，室温孵育30min，离心收集酵母细胞，并用50ml清洗液洗涤酵母3遍。用5ml清洗液重悬酵母细胞，并加入200μl SA磁珠(购自美天旎)，颠倒孵育10min。用清洗液洗涤酵母和磁珠混合物3遍，将混合物加入LS纯化柱(购自美天旎)中。将LS纯化柱放在磁力架上，用清洗液洗涤去除非特异性结合的酵母细胞。将纯化柱从磁力架上取出，加入清洗液洗脱酵母。洗脱下来的酵母离心后转入200ml SD-CAA扩增培养基中进行扩增。
流式细胞分选获得高亲和力酵母细胞
将经过MACS富集的酵母细胞接种于SD-CAA扩增培养基中，起始扩增浓度＝0.5OD 600/ml。30℃，225rpm摇瓶培养过夜。用SD-CAA诱导培养基(1L SD-CAA诱导培养基中含6.7g YNB、5g酪氨基酸、13.62g Na 2HPO 4·12H 2O、7.44g NaH 2PO 4及2％半乳糖、2％棉子糖和0.1％葡萄糖)重悬酵母细胞，起始浓度为0.5OD 600/ml，诱导过夜。加入1:200稀释的抗-c-Myc鼠源抗体(购自Thermo)和100nM生物素标记的PD-L2抗原，室温孵育10min。加入PBS清洗酵母3遍，加入1:500稀释的羊抗小鼠IgG(H+L)Alexa Fluor Plus 488荧光抗体(购自Invitrogen)和链霉亲和素APC结合物荧光抗体(购自Invitrogen)，4℃避光孵育15min。加入2ml PBS重悬细胞，使用BD FACSAriaII仪器进行分选获得可与PD-L2抗原有较高结合能力的酵母。
PD-L2纳米抗体候选分子抗体基因的调取
通过MACS和FACS富集得到的能与PD-L2抗原有较高结合能力的酵母菌液，在SD-CAA扩增培养基中30℃，225rpm培养过夜，按照酵母质粒抽提试剂盒(购自天根)操作抽提酵母质粒。质粒通过电转化入Top10感受态细胞(购自天根)，涂布氨苄抗性平板，于37℃培养过夜。挑取单克隆测序获得VHH基因序列。
1.3重链抗体的构建及表达纯化
抗体基因构建入pCDNA3.1表达载体
将VHH基因序列和人IgG1(LALA突变)Fc段相连，利用同源重组酶构建在(购自Vazyme)和EcoR I/Not I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃ 培养过夜，挑取单克隆测序，并抽提质粒。
细胞转染及蛋白纯化
采用ExpiCHO TM表达***试剂盒(购自Thermo)，将抽提质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M柠檬酸钠,pH3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀，获得目的蛋白。
1.4纯化抗PD-L2抗体与人PD-L2结合
通过转染克隆人PD-L2cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L2的CHO细胞(CHO-hPD-L2细胞)。将扩大培养的CHO-hPD-L2细胞调整细胞密度至2×10 6个细胞/ml，100μl/孔加入96孔流式板，离心备用。将纯化的PD-L2抗体用PBS稀释，1000nM开始3倍稀释共12个点，将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam),4℃孵育30min,PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中，实验结果如图1所示，本发明所有的纯化样品和CHO-hPD-L2细胞均有结合活性。
1.5 PD-L2抗体亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于高通量法的抗体-抗原亲和力和表位结合的测定.MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的PD-L2抗原中作用5min，之后将传感器转移至PBS中解离5min。使用1:1结合 模型进行动力学的分析。
表4.候选分子亲和力
编号 KD(M) Kon(1/Ms) Koff(1/s)
D-Na-58 4.86E-10 6.68E+05 3.25E-04
D-Na-64 5.66E-10 6.52E+05 3.69E-04
D-Na-67 3.58E-09 9.33E+04 3.34E-04
D-Na-78 5.95E-09 5.81E+04 3.45E-04
D-Na-80 3.40E-09 1.27E+05 4.33E-04
D-Na-87 6.55E-10 4.40E+05 2.88E-04
D-Na-89 3.31E-09 3.39E+05 1.12E-03
D-Na-90 3.79E-09 4.63E+05 1.76E-03
D-Na-96 1.27E-09 8.62E+05 1.10E-03
D-Ye-10 5.22E-09 2.03E+05 1.06E-03
D-Ye-22 4.51E-09 1.83E+05 8.25E-04
D-Ye-29 1.93E-09 1.95E+05 3.76E-04
D-Ye-31 5.04E-09 1.28E+05 6.44E-04
D-Ye-32 5.69E-09 2.56E+05 1.46E-03
1.6纯化抗PD-L2抗体阻断PD-L2与PD-1结合
通过转染克隆人PD-1cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-1的CHO细胞(CHO-hPD-1细胞)。将扩大培养的CHO-hPD-1细胞调整细胞密度至2×10 6个细胞/ml,100μl/孔加入96孔流式板，离心备用。将纯化的突变样品用PBS稀释，1000nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入生物素化人PD-L2蛋白(购自AcroBiosystems)，终浓度为1μg/ml，与纯化样品4℃孵育30min。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min,PBS清洗两次。100μl/孔加入用PBS稀释100倍的APC羊抗小鼠IgG(最小x反应性)抗体(购自Biolegend)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中，实验结果如图2所示，本发明所有的纯化样品均可以阻断PD-L2与PD-1的结合。
1.7 PD-L2抗体人源化构建
为了减少单克隆抗体在人体内产生免疫原性，将D-NA-96和D-Ye-29抗体进行人源化。人源化方法采用VHH人源化通用框架移植法，同时根据文献(Vincke,C.,等人，人源化骆驼单域抗体和鉴定通用人源化纳米抗体支架的一般策略.J Biol Chem 284(5):3273-3284)报道的方法对抗体框架2(framework2)的部分氨基酸进行突变完成。
本研究利用IMGT(http://www.imgt.org)对D-NA-96、D-Ye-29及人源化序列进行了人源化水平评估，结果如表5所示，所有人源化后的样品人源化水平均高于80％，符合后期药物开发要求。
表5.D-NA-96/D-Ye-29人源化序列和人的同源性
编号 种系 同源性
D-Na-96 IGHV3-48*03 69.40％
HZ-D-Na-96-1 IGHV3-48*03 84.70％
HZ-D-Na-96-2 IGHV3-48*03 83.70％
HZ-D-Na-96-3 IGHV3-48*03 80.60％
D-Ye-29 IGHV3-23*01 71.1％
HZ-D-Ye-29-3 IGHV3-23*01 80.40％
蛋白构建及表达纯化方法同实施例1.3，利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na 2HPO 4·12H 2O,pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20min，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm,3μm)。
表6.D-Na-96/D-Ye-29人源化抗体的纯度检测结果
编号 单体比例(％)
D-Na-96 100
HZ-D-Na-96-1 96.53
HZ-D-Na-96-2 99.92
HZ-D-Na-96-3 98.84
D-Ye-29 100
HZ-D-Ye-29-3 98.80
1.8 D-Na-96人源化样品与人PD-L2结合
本实验检测纯化获得的D-Na-96人源化样品与CHO-hPD-L2细胞结合活性，实验方法同实施例1.4，实验结果如图3所示，D-Na-96人源化样品与CHO-hPD-L2细胞有很好的结合活性，水平与D-Na-96相当。
1.9 D-Na-96/D-Ye-29人源化样品亲和力测定
本实验检测纯化获得的D-Na-96/D-Ye-29人源化样品与人PD-L2结合亲和力，实验方法同实施例1.5，实验结果如表7所示，D-Na-96/D-Ye-29人源化样品与人PD-L2蛋白有很好的结合活性。
表7.D-Na-96/D-Ye-29人源化样品亲和力
编号 KD(M) kon(1/Ms) kdis(1/s)
D-Na-96 2.50E-09 2.62E+05 6.55E-04
HZ-D-Na-96-1 1.68E-09 3.44E+05 5.77E-04
HZ-D-Na-96-2 1.58E-09 3.23E+05 5.12E-04
HZ-D-Na-96-3 1.99E-09 2.97E+05 5.90E-04
D-Ye-29 1.93E-09 1.95E+05 3.76E-04
HZ-D-Ye-29-3 4.49E-09 3.86E+05 1.73E-03
1.10 D-Na-96人源化样品阻断PD-L2与PD-1结合
本实验检测纯化获得的D-Na-96人源化样品阻断PD-L2与PD-1的结合，实验方法同实施例1.6，实验结果如图4所示，本发明所有的人源化样品均可以阻断PD-L2与PD-1的结合，且阻断水平与D-Na-96相当。
实施例2：抗人PD-L1纳米抗体的开发
2.1纳米抗体文库的构建
动物免疫
将l mg人PD-L1抗原(购自AcroBiosystems)与弗氏佐剂等体积混合，免疫2只羊驼(Llama及Alpaca各一只)，分别于第1、2、3、5、7周免疫动物，剌激B细胞表达抗原特异性的纳米抗体。5次免疫结束后，提取300ml羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼总cDNA。
其他纳米文库构建方法同实施例1.1。
2.2 PD-L1纳米抗体的筛选及重链抗体的构建及表达纯化
本研究从实施例2.1构建的酵母展示文库中，筛选出了可以和人PD-L1特异性结合的纳米抗体序列，具体筛选方法同实施例1.2。将VHH基因序列和人 IgG1(LALA突变)Fc段相连，并构建入真核表达载体pCDNA3.1中。利用ExpiCHO表达***和磁珠亲和纯化***制备了纯度较高的重链抗体蛋白。重链抗体对的构建及表达纯化方法同实施例1.3。
2.3 PD-L1抗体亲和力测定
本研究利用ForteBio仪器对获得的抗PD-L1抗体和人PD-L1蛋白的结合活性进行了检测，检测方法同实施例1.5。检测结果如表8所示，本研究获得的3个候选分子和人PD-L1蛋白均有较好的结合活性。
表8.候选分子亲和力
编号 KD(M) Kon(1/Ms) Koff(1/s)
K-Yr-13&14-02 9.60E-10 5.85E+05 5.61E-04
K-Yr-13&14-09 2.56E-09 5.35E+05 1.37E-03
K-Yr-13&14-16 1.10E-08 5.53E+05 6.06E-03
ATE 1.30E-09 4.64E+05 6.04E-04
2.4 PD-L1抗体人源化构建
为了减少单克隆抗体在人体内产生免疫原性，将K-Yr-13&14-02抗体进行人源化。人源化方法同实施例1.7。
本研究利用IMGT(http://www.imgt.org)对K-Yr-13&14-02及人源化序列进行了人源化水平评估，结果如表9所示，人源化水平均高于80％，符合后期药物开发要求。
表9.K-Yr-13&14-02人源化序列和人的同源性
编号 种系 同源性
K-Yr-13&14-02 IGHV3-11*05 74.20％
HZ-K-Yr-13&14-02-3 IGHV3-11*05 80.40％
蛋白构建及表达纯化及HPLC纯度检测方法同实施例1.3。结果如表10所示，一步纯化后获得纯度较高的人源化抗PD-L1重链抗体蛋白。
表10.HZ-K-Yr-13&14-02-3的纯度检测结果
编号 单体比例(％)
HZ-K-Yr-13&14-02-3 97.57
2.5人源化抗PD-L1纳米抗体与人PD-L1结合
本实验检测纯化获得的人源化样品HZ-K-Yr-13&14-02-3与CHO-hPD-L1细胞结合活性，实验方法同实施例1.4，实验结果如图5所示，HZ-K-Yr-13&14-02-3与CHO-hPD-L1细胞有很好的结合活性，水平与K-Yr-13&14-02及对照抗体ATE相当。
2.6人源化抗PD-L1纳米抗体亲和力测定
本实验检测纯化获得的HZ-K-Yr-13&14-02-3与人PD-L1结合亲和力，实验方法同实施例1.5，实验结果如表11所示，HZ-K-Yr-13&14-02-3与人PD-L2蛋白有很好的结合活性。
表11.人源化样品亲和力
编号 KD(M) kon(1/Ms) kdis(1/s)
HZ-K-Yr-13&14-02-3 2.23E-10 4.45E+05 9.92E-05
ATE 1.16E-09 4.37E+05 5.07E-04
实施例3抗PD-L1/PD-L2双特异性抗体
3.1抗PD-L1/PD-L2双特异性抗体的分子构建
本研究构建了3种不同形式的抗PD-L1/PD-L2双特异性抗体，其结构示意图如图6所示。
Bi-201含有一条肽链，具有SEQ ID NO:23所示的氨基酸序列，其包含抗PD-L1的纳米抗体HZ-K-Yr-13&14-02-3，所述纳米抗体氨基酸序列C端和人IgG1(LALA突变型)结构域直接相连。抗PD-L2的纳米抗体HZ-D-NA-96-1通过一段柔性肽链(GGGGSGGGGSGGGGSGGGGSG)(SEQ ID NO：27)和Fc的C端相连接。
Bi-202含有一条肽链，具有SEQ ID NO:24所示的氨基酸序列，抗PD-L1的纳米抗体HZ-K-13&14-02-03氨基酸序列C端和抗PD-L2的纳米抗体HZ-D-NA-96-1通过一段柔性肽链(GGGGSGGGGSGGGGSGGGGSG)(SEQ ID NO:27)连接。HZ-D-NA-96-1的C端和人IgG1(LALA突变型)结构域直接相连。
Bi-203-204含有两条肽链。肽链#1具有SEQ ID NO:25所示的氨基酸序列，抗PD-L1的纳米抗体HZ-K-Yr-13&14-02-3氨基酸序列C端和衍生自人IgG1的SEQ ID NO:29所示CH1氨基酸序列直接连接；将人IgG1(LALA突变型)Fc(SEQ ID NO：)结构域直接和CH1区C端连接，由此获得肽链#1。肽链#2具有SEQ ID NO:26所示的氨基酸序列，其包含抗PD-L2纳米抗体HZ-D-NA-96-01氨基酸序列SEQ ID NO:16，所述纳米抗体氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序 列SEQ ID NO:30，由此获得肽链#2。
3.2抗PD-L1/PD-L2双特异性抗体的表达和纯化
在本实施例中，将编码实施例3.1中构建的抗PD-L1/PD-L2双特异性抗体Bi-201、Bi-202、Bi-203-204的核苷酸序列均通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)。利用ExpiCHO表达***和磁珠亲和纯化***制备了纯度较高的重链抗体蛋白。蛋白构建及表达纯化及HPLC纯度检测方法同实施例1.3。结果如表12所示，一步纯化后获得纯度较高的双特异性抗体蛋白。
表12.抗PD-L1/PD-L2双特异性抗体的纯度检测结果
编号 单体比例(％)
Bi-201 98.26
Bi-202 97.67
Bi-203-204 99.26
3.3抗PD-L1/PD-L2双特异性抗体亲和力测定
本研究利用ForteBio仪器对获得的抗PD-L1/PD-L2双特异性抗体和人PD-L1蛋白或人PD-L2的结合活性进行了检测，检测方法同实施例1.5。检测结果如表13、表14所示，本研究获得的3个候选分子和人PD-L1及人PD-L2蛋白均有较好的结合活性。
表13.候选分子和人PD-L1蛋白的亲和力
编号 KD(M) Kon(1/Ms) Koff(1/s)
Bi-201 4.81E-10 3.34E+05 1.61E-04
Bi-202 1.66E-09 2.07E+05 3.43E-04
Bi-203-204 1.59E-09 2.54E+05 4.05E-04
HZ-K-Yr-13&14-02-3 1.38E-09 2.24E+05 3.09E-04
ATE 7.97E-09 2.69E+05 2.15E-03
表14.候选分子和人PD-L2蛋白的亲和力
编号 KD(M) Kon(1/Ms) Koff(1/s)
Bi-201 4.45E-10 5.25E+05 2.34E-04
Bi-202 1.20E-09 3.29E+05 3.95E-04
Bi-203-204 1.14E-09 3.72E+05 4.24E-04
HZ-D-NA-96-1 1.11E-09 3.60E+05 4.00E-04
3.4抗PD-L1/PD-L2双特异性抗体与细胞表面人PD-L1或人PD-L2的结合
本实验检测纯化获得的抗PD-L1/PD-L2双特异性抗体与CHO-hPD-L1细胞或CHO-hPD-L2结合活性，实验方法同实施例1.4，实验结果如图7A和7B所示，Bi-201、Bi-202、Bi-203-204与CHO-hPD-L1细胞及CHO-hPD-L2细胞均有很好的结合活性。
3.5抗PD-L1/PD-L2双特异性抗体阻断PD-L2/PD-L1与PD-1结合
将扩大培养的CHO-hPD-1细胞调整细胞密度至2×10 6个细胞/ml，100μl/孔加入96孔流式板，离心备用。将纯化的突变样品用PBS稀释，1000nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入生物素化人PD-L2蛋白或生物素化人PD-L1蛋白(购自AcroBiosystems)，终浓度为1μg/ml，与纯化样品4℃孵育30min。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min,PBS清洗两次。100μl/孔加入用PBS稀释100倍的APC羊抗小鼠IgG(最小值x反应性)抗体(购自Biolegend)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中，实验结果如图8A和8B所示，本发明所有的纯化的双特异性抗体样品均可以阻断PD-L2及PD-L1与PD-1的结合。
3.6抗PD-L1/PD-L2双特异性抗体阻断PDL1/PDL2/PD1/luc信号通路实验
PD-L1和PD-L2能够共表达于肿瘤细胞或免疫细胞上，本实施例利用共表达人PD-L1和人PD-L2的CHO细胞(CHO-K1-PD-L1/PD-L2)和过表达人PD-1且含有NFAT-荧光素酶报告基因的Jurkat细胞(Jurkat-PD-1-NFAT)共孵育的方法检测了纯化抗体Bi-201、Bi-202、Bi-203-204对PD-L1/PD-1通路和PD-L2/PD-1通路的同时阻断作用，具体方法如下。
将CHO-K1-PD-L1/PD-L2细胞调整密度为5×10 5细胞/ml，100μl/孔接种于96孔细胞培养白底板，置于37℃，5％CO 2培养箱培养过夜。将纯化抗体和对照抗体1640完全培养基梯度稀释后，备用。将Jurkat-PD-1-NFAT细胞用1640完全培养基调整细胞密度至2.5×10 5细胞/ml，备用。取出白底板，吸去培养上清，将上述样品稀释好的至对应浓度，取40μl/孔加入白底板，同时加入40μl/孔Jurkat-PD-1-NFAT效应细胞悬液，置于37℃，5％CO 2培养箱贴壁培 养6小时。向每孔中加入Bio-Glo TM试剂(Promega)，使用多功能酶标仪读取荧光信号值。
实验结果如图9所示。本发明的抗PD-L1/PD-L2双特异性抗体可以在体外同时阻断PD-L1/PD-1和PD-L2/PD-1信号通路，激活下游报告基因的表达。而抗PD-L1或抗PD-L2单抗均无法完全阻断该通路并激活下游报告基因的表达。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种抗PD-L2纳米抗体，其特征在于，所述PD-L2纳米抗体的VHH链的各互补决定区CDR由以下组成：
    氨基酸序列如SEQ ID NO:57所示的CDR1；氨基酸序列如SEQ ID NO:58所示的CDR2；和氨基酸序列如SEQ ID NO:59所示的CDR3；或
    氨基酸序列如SEQ ID NO:60所示的CDR1；氨基酸序列如SEQ ID NO:61所示的CDR2；和氨基酸序列如SEQ ID NO:62所示的CDR3；
    或者，所述抗PD-L2纳米抗体的VHH链的氨基酸序列如SEQ ID NO:1、2、4、5、6、7、8、9、10、11、13、14、17或18所示。
  2. 一种抗PD-L1纳米抗体，其特征在于，所述PD-L1纳米抗体的VHH链的各互补决定区CDR由以下组成：
    氨基酸序列如SEQ ID NO:63所示的CDR1；氨基酸序列如SEQ ID NO:64所示的CDR2；和氨基酸序列如SEQ ID NO:65所示的CDR3。
  3. 一种双特异性抗体，其特征在于，所述双特异性抗体包括：如权利要求2所述的抗PD-L1纳米抗体和如权利要求1所述的抗PD-L2纳米抗体。
  4. 如权利要求1所述的双特异性抗体，其特征在于，所述的双特异性抗体含有如式I或式II所示结构的多肽，或同时含有如式III和式IV所示结构的多肽，
    A-L1-Fc1-L2-B  (式I)
    A-L3-B-L4-Fc1  (式II)
    A-L5-Fc2-L6-Fc1  (式III)
    B-L7-Fc2  (式IV)
    其中，
    A和B各自独立地为如权利要求2所述的抗PD-L1纳米抗体或如权利要求1所述的抗PD-L2纳米抗体，且A和B是不同的抗体；
    L1、L2、L3和L4各自独立地为肽键或接头元件；
    Fc1、Fc2均为抗体的Fc段；其中Fc1为人IgG结构域(优选地为LALA突变型IgG结构域)，并且Fc2为CH1+CL结构域；
    “-”为肽键。
  5. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述 的抗PD-L2纳米抗体、如权利要求2所述的抗PD-L1纳米抗体，或如权利要求3所述的双特异性抗体。
  6. 一种载体，其特征在于，所述载体含有如权利要求5所述的多核苷酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求6所述的载体，或其基因组中整合有如权利要求5所述的多核苷酸；
    或者，所述的宿主细胞表达如权利要求1所述的抗PD-L2纳米抗体、如权利要求2所述的抗PD-L1纳米抗体，或如权利要求3所述的双特异性抗体。
  8. 一种产生双特异性抗体的方法，其特征在于，包括步骤：
    (a)在合适的条件下，培养如权利要求7所述的宿主细胞，从而获得含所述抗PD-L2纳米抗体、抗PD-L1纳米抗体，或双特异性抗体的培养物；和
    (b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的抗PD-L2纳米抗体、抗PD-L1纳米抗体，或双特异性抗体。
  9. 一种免疫偶联物，其特征在于，所述免疫偶联物含有：
    (a)如权利要求1所述的抗PD-L2纳米抗体、如权利要求2所述的抗PD-L1纳米抗体，或如权利要求3所述的双特异性抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
  10. 如权利要求1所述的抗PD-L2纳米抗体、如权利要求2所述的抗PD-L1纳米抗体，或如权利要求3所述的双特异性抗体，或如权利要求9所述的免疫偶联物的用途，其特征在于，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1和/或PD-L2；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤或是表达PD-L2的肿瘤。
  11. 一种药物组合物，其特征在于，含有：(i)如权利要求1所述的抗PD-L2纳米抗体、如权利要求2所述的抗PD-L1纳米抗体，或如权利要求3所述的双特异性抗体，或如权利要求9所述的免疫偶联物；以及(ii)药学上可接受的载体。
  12. 一种PD-L1和/或PD-L2检测试剂，其特征在于，所述的检测试剂包含如权利要求9所述的免疫偶联物和检测学上可接受的载体。
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