WO2022194153A1 - Pd-l1和tlr7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 - Google Patents

Pd-l1和tlr7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 Download PDF

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WO2022194153A1
WO2022194153A1 PCT/CN2022/080953 CN2022080953W WO2022194153A1 WO 2022194153 A1 WO2022194153 A1 WO 2022194153A1 CN 2022080953 W CN2022080953 W CN 2022080953W WO 2022194153 A1 WO2022194153 A1 WO 2022194153A1
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seq
antibody
nanobody
cdr2
cdr1
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French (fr)
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宫丽崑
尉骁璐
龙益如
孙建华
童永亮
刘婷婷
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中国科学院上海药物研究所
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Definitions

  • the invention relates to the field of biomedicine, and more particularly to a PD-L1 and TLR7 double-targeted nanobody conjugated drug and its application in anti-tumor.
  • Tumors achieve immune escape by upregulating the expression of immune checkpoint molecules such as PD-L1.
  • immune checkpoint molecules such as PD-L1.
  • a variety of antibody drugs have been developed for the treatment of tumors against immune checkpoint molecular targets such as PD-L1.
  • immune checkpoint blockade therapy such as PD-L1 antibodies still faces problems such as low patient response rates.
  • Combination therapy is an effective way to improve the anti-tumor efficacy and patient response rate of immune checkpoint blockade therapy.
  • scientifically reasonable, safe and effective combination therapy still needs to be further studied.
  • TLRs Toll-like receptors
  • TLR7 agonists have been widely studied and applied in anti-tumor therapy.
  • TLR agonists are one of the potential combination targets for immune checkpoint blockade therapy.
  • Nanobodies are currently the smallest antibody molecules. In addition to the specificity of monoclonal antibodies, nanobodies have the advantages of strong tissue penetration, low immunogenicity, good stability, simple humanization, and easy preparation. Nanobody drug conjugates developed based on nanobodies are a novel form of drug molecules, which have broad application prospects in the fields of drug delivery, in vivo imaging, and anti-tumor therapy.
  • the purpose of the present invention is to provide a novel and effective PD-L1 nanobody conjugated drug.
  • the purpose of the present invention is to provide a combination of PD-L1 nanobody and TLR7 agonist for anti-tumor therapy and to provide a PD-L1 and TLR7 double-targeted nanobody conjugated drug.
  • Another object of the present invention is to provide the application of PD-L1 and TLR7 dual-targeted nanobody conjugated drug in tumor prevention and treatment, especially in tumors with low response rate of PD-L1 antibody.
  • an antibody-drug conjugate or a pharmaceutically acceptable salt thereof is provided, and the structure of the antibody-drug conjugate is shown in formula I:
  • Ab is PD-L1 antibody
  • U is each independently a TLR agonist
  • J is a chemical bond or linker
  • n 0 or a positive integer
  • the PD-L1 antibody includes monospecific antibody, bispecific antibody, and multispecific antibody (eg, trispecific antibody).
  • the PD-L1 antibody includes: monoclonal antibody, single-chain antibody (scFv), and nanobody.
  • the PD-L1 antibody includes a monovalent, bivalent or multivalent antibody.
  • the PD-L1 antibody includes an antibody in the form of a multimer.
  • the PD-L1 antibody specifically binds to PD-L1.
  • the PD-L1 antibody includes a PD-L1 monovalent nanobody, a bivalent nanobody and/or a multivalent nanobody.
  • the PD-L1 antibody includes a blocking type (which can block the binding of PD-L1 and PD-1), a non-blocking type (which does not block the binding of PD-L1 and PD-1) ), or a combination thereof.
  • the PD-L1 antibody is a blocking antibody.
  • the PD-L1 antibody blocks the binding of PD-1 to PD-L1.
  • the PD-L1 is human PD-L1 or non-human mammalian PD-L1 (eg mouse PD-L1).
  • the PD-L1 antibody is a human or non-human mammalian antibody.
  • the non-human mammal is selected from the group consisting of camel, alpaca, mouse, and cynomolgus monkey.
  • the PD-L1 antibody is a PD-L1 nanobody or a derivative antibody thereof.
  • the derivatized antibody is a modification of the PD-L1 nanobody, including but not limited to linking the PD-L1 nanobody to an Fc fragment, human serum albumin, polyethylene glycol PEG, forming two valent antibodies and/or multivalent antibodies.
  • the nanobodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.
  • the PD-L1 Nanobody specifically binds to PD-L1, and the complementarity determining region CDR of the VHH chain in the Nanobody is selected from one or more of the following groups:
  • the PD-L1 Nanobody specifically binds to human PD-L1, and the complementarity determining region CDRs of the VHH chain in the Nanobody are selected from one or more of the following groups:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the ability to bind to PD-L1.
  • amino acid sequence of the VHH chain of the anti-PD-L1 Nanobody is selected from the following group:
  • (a) has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:17;
  • the Nanobody sequence comprises at least 80% with SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, Preferably amino acid sequences of at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity.
  • the anti-PD-L1 nanobody further includes a nanobody that specifically binds to human PD-L1.
  • amino acid sequence of the VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the following group:
  • (a) has the amino acid sequence shown in SEQ ID NO: 21, 25, 29, 33, 37, 40, 44, 48, 52, 56, 59;
  • the Nanobody sequence comprises at least 80%, preferably at least 90% with SEQ ID NO: 21, 25, 29, 33, 37, 40, 44, 48, 52, 56 or 59 , more preferably at least 95%, even more preferably at least 99% sequence similarity amino acid sequences.
  • the anti-PD-L1 nanobody further includes a humanized nanobody that specifically binds to human PD-L1.
  • amino acid sequence of the humanized specific binding to the VHH chain of the human PD-L1 Nanobody is selected from the following group:
  • the Nanobody sequence comprises and SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91 have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% % sequence similarity to amino acid sequences.
  • the anti-PD-L1 nanobody further comprises an affinity matured specific binding human PD-L1 nanobody.
  • amino acid sequence of the affinity-matured VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the group consisting of:
  • the "affinity maturation” refers to that the affinity of the anti-human PD-L1 Nanobody modified by affinity maturation for PD-L1 is relative to that of the anti-human PD-L1 Nanobody before modification to PD-L1
  • the affinity is increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12 times, at least 20 times, or at least 25 times.
  • the Nanobody sequence comprises and SEQ ID NO: 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 , 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity amino acid sequence.
  • the CDRs of the CDRs of the VHH chain of the Nanobody consist of CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4 .
  • the VHH chain sequence of the Nanobody is shown in SEQ ID NO.: 1.
  • the TLR agonist is a macromolecule (protein or nucleic acid) or small molecule agonist.
  • the TLR agonists include but are not limited to TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists agent.
  • n is the average number of drugs conjugated in the antibody-drug conjugate, preferably n is 1-9, preferably 2.5-6.5, more preferably 3.5-5.5.
  • the TLR agonist is a TLR7 agonist.
  • the TLR agonist does not have TLR8 agonistic activity.
  • the TLR7 agonist is a host endogenous agonist or an exogenous agonist.
  • the TLR7 agonist is a small molecule agonist.
  • the TLR7 agonist includes: SZU-101:
  • the TLR7 agonist is a derivative compound of SZU-101, including but not limited to the substitution, modification or deletion of one or more groups on the basis of SZU-101.
  • the TLR7 agonist is a multivalent compound of SZU-101.
  • the TLR7 agonist (such as SZU-101) is linked to the terminal amino group or the side chain amino group of the heavy chain constant region or heavy chain variable domain (VHH) of the PD-L1 antibody.
  • the TLR7 agonist (eg SZU-101) is linked to the sulfhydryl group of the PD-L1 antibody.
  • the SZU-101 is linked to the amino group of the PD-L1 antibody, and forms the structure shown by S1:
  • the SZU-101 is connected to the sulfhydryl group of the PD-L1 antibody and forms the structure shown in S2:
  • the TLR7 agonist is site-directed and/or randomly linked to the PD-L1 antibody (that is, in formula I, the U is site-directed and/or randomly linked to Z).
  • the U is connected to Z at a fixed point.
  • the U is site-specifically linked to an amino acid site of the PD-L1 antibody Z selected from the group consisting of G, K, L, A, C or a combination thereof.
  • the chemical bond is polyethylene glycol PEG.
  • the chemical bond is a derivative compound of PEG, including but not limited to the substitution, modification or deletion of one or more groups on the basis of SZU-101.
  • the degree of polymerization of the PEG chemical bond is a positive integer greater than or equal to 1.
  • the antibody-drug conjugate increases the level of PD-L1 in intratumoral cells.
  • the antibody-drug conjugate activates immune cells.
  • the activation is in vitro activation.
  • the in vitro activation comprises: in the presence of the antibody-drug conjugate, culturing the immune cells for a period of time (eg, 6-48 hours), so as to obtain immune-activated immunity cell.
  • the immune cells are selected from but not limited to: CD8+ T cells, natural killer cells NK, dendritic cells, lymphocytes, monocytes/macrophages, granulocytes, or a combination thereof.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is used to prepare a composition or formulation, and the composition or formulation is used for:
  • the remodeling of the tumor immune microenvironment is the coordination of intratumoral innate immunity and adaptive immunity against tumor immune responses.
  • the remodeling of the tumor immune microenvironment is to increase the infiltration of anti-tumor immune cells and reduce the proportion of immunosuppressive cells.
  • the anti-tumor immune cells include but are not limited to CD8+ T cells and NK cells that secrete granzyme and IFN- ⁇ , activated dendritic cells, and CD4+ T cells that secrete IFN- ⁇ , M1 macrophages.
  • the immunosuppressive cells include, but are not limited to, M2 macrophages, Treg cells, and leukocytes that secrete TGF- ⁇ .
  • the PD-L1 level includes cell surface PD-L1 level and intracellular PD-L1 level.
  • the tumor with low PD-L1 expression is a solid tumor or a hematological tumor.
  • composition comprising:
  • the pharmaceutical composition further comprises:
  • the other biologically active drugs promote the anti-tumor function of CD8+ T cells and NK cells.
  • the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.
  • the administration mode of the pharmaceutical composition is selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spray and mist inhalation.
  • the pharmaceutical composition is administered by combining the pharmaceutical composition with immune cells (such as dendritic cells, natural killer cells, lymphocytes, monocytes/macrophages, granulocytes, etc. ) after co-cultivation, the immune cells were isolated and reinfused in vivo.
  • immune cells such as dendritic cells, natural killer cells, lymphocytes, monocytes/macrophages, granulocytes, etc.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid state, solid state, or gel state.
  • the pharmaceutical composition is used for antitumor therapy.
  • the pharmaceutical composition is used to treat tumors with low PD-L1 expression.
  • low expression of PD-L1 means that the amount E1 of PD-L1 expressed by the tumor is lower than the amount E0 of PD-L1 expressed by normal tumors, preferably E1/E0 ⁇ 1/2, More preferably ⁇ 1/3, more preferably ⁇ 1/4.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small bowel cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, lymphatic cancer, bone cancer, adrenal tumor, or bladder tumor.
  • an immunoconjugate is provided, and the immunoconjugate contains:
  • the other coupling moieties are selected from the group consisting of small molecule compounds, PEG, fluorescein, radioisotopes, contrast agents, fatty acid chains, protein fragments, or combinations thereof.
  • the components (a) and (b) are operably linked.
  • the coupling moiety includes chemical markers and biological markers.
  • the chemical label is selected from isotopes, immunotoxins and/or chemical drugs.
  • the biomarker is selected from biotin, avidin or enzyme label.
  • the small molecule compound is selected from drugs or toxins for the treatment of tumors or autoimmune diseases.
  • the radioisotope includes:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.
  • the radioisotopes include but are not limited to iodine-131, indium-111 and lutetium-177.
  • the contrast agent is used for MRI or CT.
  • the protein fragments include but are not limited to antibody Fc, biotin, avidin, HRP, antibodies, enzymes, cytokines and other biologically active proteins or polypeptides.
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing Enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles that can detect products , prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • a fusion protein comprises:
  • the polypeptide molecules or fragments with therapeutic function include but are not limited to: targeting PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB, CD47, TIM3, Polypeptide molecules or fragments of CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2.
  • polypeptide molecules or fragments with therapeutic function include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cells factor.
  • polypeptide molecule or fragment with therapeutic function includes single-chain antibody (scFv), diabody, monoclonal antibody, or chimeric antibody.
  • the fusion protein further comprises a tag sequence that facilitates expression and/or purification.
  • the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.
  • the fusion protein includes bispecific antibody and chimeric antibody.
  • a multispecific antibody comprising:
  • the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB , CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2, or a combination thereof.
  • a target selected from the group consisting of PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB , CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2, or a combination thereof.
  • the second antigen-binding region is a nanobody.
  • the multispecific antibody includes one or more second antigen binding regions.
  • the multispecific antibody further comprises the Fc segment of the antibody.
  • a reaction system is configured, the reaction system includes an antibody and a free drug molecule, and then a coupling reaction is performed to prepare the antibody-drug conjugate, wherein the drug molecule includes a TLR agonist and a linker.
  • reaction time is 3h-10h.
  • the molar ratio of the antibody to the drug molecule is 1-2:3-20; preferably 1:6-10.
  • SZU-101, EDCI and NHS were dissolved in DMSO and stirred for three hours at room temperature to prepare SZU-101-NHS active ester, PD-L1 nanobody and SZU-101-NHS active ester were 1:10
  • the molar ratio dose was stirred at 4 °C for 4 hours, and the nanobody conjugated drug was prepared.
  • a method for preventing or treating tumors is provided, by administering the nanobody conjugated drug as described in the first aspect of the present invention to a subject in need.
  • the tumor is a tumor expressing PD-L1.
  • the tumor is selected from the group consisting of tumors with high PD-L1 expression, tumors with moderate PD-L1 expression, and tumors with low PD-L1 expression.
  • the tumor is a tumor that expresses moderate PD-L1 or a tumor that expresses low PD-L1.
  • the tumor is a tumor with low expression of PD-L1.
  • "highly expressing PD-L1" means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0)>1, more preferably ⁇ 1.5, more preferably ⁇ 2.0.
  • "moderately expressing PD-L1" means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) is 0.5-1.1, more It is preferably 0.7-1.0, more preferably 0.8-0.9.
  • low expression of PD-L1 means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) ⁇ 1/2, more Preferably ⁇ 1/3, more preferably ⁇ 1/4.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small bowel cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, lymphatic cancer, bone cancer, adrenal tumor, or bladder tumor.
  • a PD-L1 Nanobody specifically binds to PD-L1, and the CDR of the VHH chain in the Nanobody is selected from the group consisting of one or more of:
  • the PD-L1 Nanobody specifically binds to PD-L1, and the complementarity determining region CDR of the VHH chain in the Nanobody is selected from one or more of the following groups:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the ability to bind to PD-L1.
  • amino acid sequence of the VHH chain of the anti-PD-L1 Nanobody is selected from the following group:
  • (a) has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:17;
  • the Nanobody sequence comprises at least 80% with SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, Preferably amino acid sequences of at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity.
  • the anti-PD-L1 nanobody further includes a nanobody that specifically binds to human PD-L1.
  • amino acid sequence of the VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the following group:
  • (a) has the amino acid sequence shown in SEQ ID NO: 21, 25, 29, 33, 37, 40, 44, 48, 52, 56, 59;
  • the Nanobody sequence comprises at least 80%, preferably at least 90% with SEQ ID NO: 21, 25, 29, 33, 37, 40, 44, 48, 52, 56 or 59 , more preferably at least 95%, even more preferably at least 99% sequence similarity amino acid sequences.
  • the anti-PD-L1 nanobody further includes a humanized nanobody that specifically binds to human PD-L1.
  • amino acid sequence of the humanized specific binding to the VHH chain of the human PD-L1 Nanobody is selected from the following group:
  • the Nanobody sequence comprises and SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91 have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% % sequence similarity to amino acid sequences.
  • the anti-PD-L1 nanobody further comprises an affinity matured specific binding human PD-L1 nanobody.
  • amino acid sequence of the affinity-matured VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the group consisting of:
  • the Nanobody sequence comprises and SEQ ID NO: 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 , 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity amino acid sequence.
  • the "affinity maturation” refers to that the affinity of the anti-human PD-L1 Nanobody modified by affinity maturation for PD-L1 is relative to that of the anti-human PD-L1 Nanobody before modification to PD-L1
  • the affinity is increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12 times, at least 20 times, or at least 25 times.
  • a medicine kit is provided, and the medicine box comprises:
  • Figure 1 shows the EC50 curve of ELISA assay for the binding of Nanobody Nb16 to PD-L1.
  • Figure 2 shows the IC50 curve of the ELISA assay of nanobody Nb16 for blocking the binding of PD-1 to PD-L1.
  • Figure 3 shows the in vivo anti-tumor effect of PD-L1 nanobody and TLR7 agonist combination therapy, wherein 3A is the experimental tumor loading and administration days; 3B is the mouse tumor growth curve; 3C is the end point tumor weight; 3D is the end point tumor weight. Endpoint tumor anatomy photos.
  • Figure 4 shows the schematic structure of the PD-L1 and TLR7 dual-targeting nanobody drug conjugates (4A) and the mass spectrometry identification before and after conjugation (4B and 4C).
  • Figure 5 shows the EC50 curve of the PD-L1 and TLR7 dual-targeting nanobody conjugated drug Nb16-SZU-101 binding to PD-L1 determined by flow cytometry, in which the nanobody Nb16 is the control.
  • Figure 6 shows the IC50 curve of PD-L1 and TLR7 dual-targeting nanobody conjugated drug Nb16-SZU-101 blocking the binding of PD-1 to PD-L1 determined by flow cytometry, in which the nanobody Nb16 is the control.
  • Figure 7 shows the anti-tumor effect of PD-L1 and TLR7 dual-targeted nanobody drug conjugate Nb16-SZU-101 in the induced CT26 tumor model, where 7A is the mouse tumor growth curve; 7B is the end point tumor weight; 7C Anatomical photographs of end-point tumors.
  • Figure 8 shows the antitumor effect of PD-L1 and TLR7 dual-targeting nanobody drug conjugate Nb16-SZU-101 in uninduced and induced CT26 tumor models, where 8A is IFN- ⁇ -induced (PD-L1 high Comparison of PD-L1 expression on the surface of CT26 cells without IFN- ⁇ induction (low PD-L1 expression); 8B is the mouse tumor growth curve; 8C is the end point tumor weight; 8D is the end point tumor anatomy photo .
  • Figure 9 shows the anti-tumor effect of PD-L1 and TLR7 dual-targeted nanobody drug conjugate Nb16-SZU-101 in B16 tumor model, in which 9A is the PD-L1 expression of B16-F10 cells that did not induce low PD-L1 expression.
  • Schematic diagram of L1 expression blank is the blank control group, isotype control is the isotype control group; 9B is the mouse tumor growth curve; 9C is the end point tumor weight; 9D is the end point tumor anatomy photo.
  • Figure 10 shows the tumor suppressive effect of PD-L1 and TLR7 dual-targeting nanobody drug conjugate Nb16-SZU-101 in early CT26 model (tumor volume ⁇ 50mm 3 ) and late stage model (tumor volume >200mm 3 ), where 10A is the mouse tumor growth curve in the early model, 10B is the mouse tumor survival curve in the early model; 10C is the mouse tumor growth curve in the late model, 10D is the mouse tumor survival curve in the late model; 10E is Tumor growth curves in mice in a re-tumor model.
  • Figure 11 shows that the administration of PD-L1 and TLR7 dual-targeted nanobody conjugated drug Nb16-SZU-101 remodels the tumor immune microenvironment, where total is the total cell mass, mFc is the isotype control group (mFc), and Nb16- SZU-101 is a nanobody conjugated drug group.
  • Figure 12 shows that PD-L1 and TLR7 dual-targeted nanobody drug conjugate Nb16-SZU-101 exerts anti-tumor effect through CD8+ T cells and NK cells when administered.
  • 12A is the tumor growth curve
  • 12B is the end point tumor weight
  • 12C is the end point tumor photo.
  • Figure 13 shows the human PD-L1 binding activity of candidate anti-human PD-L1 Nanobodies, wherein blank is a negative control.
  • Figure 14 shows the assay of the blocking activity of candidate anti-human PD-L1 Nanobodies on human PD-1/PD-L1 binding, wherein blank and blank+ligand are controls.
  • the inventors screened and identified a PD-L1 nanobody for the first time, and developed a PD-L1 and TLR7 dual-targeting nanobody conjugated drug. Specifically, it was found in various mouse xenograft models that the dual-targeted nanobody drug conjugate of the present invention has excellent anti-tumor activity.
  • the present invention also unexpectedly found that the dual-targeted nanobody conjugated drug of the present invention promotes the expression of PD-L1 in intratumoral macrophages, and mainly exerts anti-tumor activity in vivo through CD8+ T cells and NK cells. Beneficial for the treatment of "cold" tumors with low expression of PD-L1 molecules.
  • the PD-L1 and TLR7 double-targeted nanobody conjugated drug developed by the invention exhibits outstanding anti-tumor effect and novel action mechanism, and has clinical development and application value. The present invention has been completed on this basis.
  • TLR receptor refers to Toll-like receptors, which are an important class of innate immune pattern recognition receptors in the immune system of organisms, which can specifically recognize relatively conserved antigen molecules in the evolution of pathogenic microorganisms ( or pathogen-associated molecular patterns), to achieve efficient detection of pathogenic microbial invasion and induction of innate immune responses.
  • TLR1-TLR10 TLR1-TLR10
  • TLR3, TLR7, TLR8, and TLR9 are located on the endosome and lysosomal membranes of cells, and the rest are located on the cytoplasmic membrane.
  • TLR7 is preferably used as one of the drug molecule targets.
  • the natural ligand for TLR7 molecules is single-stranded linear RNA.
  • TLR receptor agonist refers to a macromolecule (protein or nucleic acid) or small molecule agonist that can specifically bind to and activate the TLR receptor, promote the transduction of downstream signaling of the TLR receptor, and achieve intrinsic Activation of immune cells.
  • TLR7 agonists are preferred to construct Nanobody drug conjugates.
  • available TLR7 agonists also include imiquimod, R848, and the like.
  • Nanobodies of the present invention As used herein, the terms “Nanobodies of the present invention”, “Nanobodies targeting PD-L1 of the present invention”, “Anti-PD-L1 Nanobodies of the present invention” are used interchangeably, and all refer to specific recognition and binding to Nanobodies against PD-L1 (including human or murine PD-L1). Particularly preferred is a Nanobody (Nb16) whose amino acid sequence of the VHH chain is shown in SEQ ID NO: 1.
  • the terms "drug conjugated Nanobody of the present invention”, “drug conjugate of dual-targeted Nanobody of the present invention”, “drug conjugated nanobody of the present invention with dual targeting of PD-L1 and TLR7” can be used interchangeably. Used interchangeably, both refer to new drug molecules formed by nanobodies that specifically recognize and bind to PD-L1 (including human or murine PD-L1) and their derived proteins coupled to TLR7 agonists.
  • the Nanobody in the Nanobody conjugated drug is particularly preferably a Nanobody whose amino acid sequence of the VHH chain is shown in SEQ ID NO: 1.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • single domain antibody sdAb, or VHH
  • nanobody a single domain antibody
  • VHH single domain antibody
  • VHH single domain antibody
  • Nanobody/single domain antibody (Nanobody), as a new type of small molecule antibody fragment, is cloned from the heavy chain variable region (VHH) of camelid natural heavy chain antibody.
  • VHH heavy chain variable region
  • Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15kDa, which is one tenth of that of a complete antibody. It has good tissue penetration, high specificity and good water solubility. Due to its special structural properties, it has the advantages of both traditional antibodies and small molecule drugs, and almost perfectly overcomes the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions, and has gradually become a new generation of antibody therapy in the new generation. It shows broad application prospects in immunodiagnosis and therapy.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable domains of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form the conjugate.
  • Nanobody drug conjugate and “Nanobody drug conjugate” are used interchangeably.
  • Nanobody conjugates are a special form of antibody-drug-drug conjugates that combine Nanobodies or derived proteins with drugs, toxins, cytokines, radionuclides, enzymes and other diagnostics Or the drug molecule form formed by therapeutic molecules can be used for tumor treatment, drug delivery and in vivo imaging, etc., and has broad clinical application value.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • 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.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to a polypeptide that specifically binds to PD-L1, such as a protein with a heavy chain variable region or peptide. They may or may not contain the starting methionine.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), the amino acid sequence of the four FRs It is relatively conservative and does not directly participate in the binding reaction.
  • FRs framework regions
  • These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in spatial structure, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • 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. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having PD-L1 binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the present invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the present invention also includes fragments of the Nanobodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences and various additional coding sequences for the mature polypeptides; coding sequences (and optional additional coding sequences) for the mature polypeptides and non-coding sequences .
  • 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 to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • 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; There are denaturing agents, 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 Hybridization occurs when it is above 95%. Furthermore, 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 fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: 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 polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. 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 protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. Therapeutic agents (eg, , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • ADCs Antibody-Drug Conjugates
  • the present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated.
  • the effector molecule is preferably a drug with therapeutic activity or a drug with promoting immune function.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg, ethylene glycol polymers
  • therapeutic agents eg, ethylene glycol polymers
  • Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the linker Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (eg, iodo, bromo, or chloro); haloesters (eg, iodo, bromo, or chloro) ); halogenated methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide
  • the drug can generally be any cytotoxic, cytostatic or immunosuppressive drug.
  • a drug is a drug that activates or promotes an immune response, such as activating an innate immune response to assist in the activation of adaptive immunity.
  • the drug is a TLR receptor agonist.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • Useful drug classes include, for example, TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists, such as SZU-101, Quimod, R848, CpG, etc.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise binding the antibody to a bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is PD-L1 antibody
  • U is each independently a TLR agonist
  • J is a chemical bond or linker
  • n 0 or a positive integer
  • the present invention provides uses of the antibodies of the present invention, for example, for the preparation of diagnostic preparations, or the preparation of medicaments for the prevention and/or treatment of PD-L1-related diseases.
  • the PD-L1-related diseases include inflammatory diseases, autoimmune diseases, etc., including but not limited to breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small bowel cancer, prostate cancer, colorectal cancer, prostate cancer, Cancer of the cervix, lymphoma, bone, adrenal gland, or bladder.
  • cancers that do not respond to treatment with one or more checkpoint inhibitors are referred to as cold tumor.
  • Cancers that respond to treatment with one or more checkpoint inhibitors are also referred to as warm or hot tumors.
  • Such tumors are thought to have higher tumor-infiltrating lymphocyte (TIL) levels and/or higher tumor mutational burden than tumors that do not respond to checkpoint inhibitor therapy.
  • TIL tumor-infiltrating lymphocyte
  • a preferred antibody-drug conjugate provided by the present invention also shows extremely significant anti-tumor activity and response rate in cold tumor and tumor models with low PD-L1 expression, and plays a significant role in various transplanted tumor models antitumor efficacy.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, as well as a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains the above-mentioned antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and pharmaceutically acceptable Accepted carrier or excipient.
  • a safe and effective amount eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %
  • pharmaceutically acceptable Accepted carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation 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.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration, the patient's health status, etc., which are all within the skill of the skilled physician.
  • the present invention provides a combination therapy scheme of PD-L1 nanobody and TLR7 agonist for the first time, which shows significant anti-tumor activity in vivo, indicating that PD-L1 antibody therapy and TLR7 immune agonist are rational in combination and can synergize Antitumor.
  • the present invention develops for the first time PD-L1 and TLR7 dual-targeted nanobody conjugated drugs, which can promote the up-regulation of PD-L1 expression in tumor cells, and coordinate intratumoral innate immunity and adaptive anti-tumor immune responses, so that the It has shown excellent tumor growth inhibition in a variety of tumors where PD-L1 antibody treatment is less effective, such as "cold" tumors and tumors with low PD-L1 expression.
  • the PD-L1 and TLR7 dual-targeted nanobody drug conjugates provided by the present invention for the first time can target the tumor immune microenvironment, reshape the tumor immune microenvironment, improve the infiltration of anti-tumor immune cells, and reduce the infiltration of immunosuppressive cells. infiltration.
  • the PD-L1 and TLR7 dual-targeted nanobody conjugated drug provided by the present invention for the first time has a clear anti-tumor efficacy mechanism, which mainly relies on CD8+ T cells and NK cells to play tumor killing and inhibition.
  • the mouse PD-L1(ECD)-pFUSE-hIgG1-Fc vector was constructed and transformed into DH5 ⁇ chemically competent cells. Monoclonal strains were obtained by screening on LB solid medium containing bleomycin resistance, and inoculated to extract plasmids.
  • the mouse PD-L1 protein was expressed by mammalian HEK293F cells, and the HEK293F cells were cultured in serum-free medium.
  • the PEI-based plasmid was complexed to complete the transfection. After 5 days of protein expression, the protein supernatant was collected and passed through a Protein A affinity column. purified protein.
  • Nanobody-displaying phages were obtained by adding helper phages to a nanobody library with a 10-fold capacity.
  • the 96-well microtiter plate was coated with 5 ⁇ g/mL NeutrAvidin solution (100 ⁇ L per well) at 4°C overnight. The next day, the cells were blocked with 2% nonfat dry milk at room temperature for 2 h, and washed 5 times with 20 mM HEPES (pH 7.5) and 150 mM NaCl solution.
  • the eluted phages were infected with TG1 competent in logarithmic growth phase, serially diluted and spread on plates for overnight culture.
  • 96 clones were picked and inoculated into a 96-well round bottom plate with 100 ⁇ L of medium per well and left to stand overnight as a master plate, and then 10 ⁇ L of overnight cultured bacterial solution was drawn into a 96-well deep bottom plate of 1 mL of medium per well to induce nanobodies. Expressed and crude.
  • the 96-well microtiter plate was coated with 5 ⁇ g/mL NeutrAvidin solution at 4°C, 700rpm, overnight.
  • BSA bovine serum albumin
  • the enzyme-labeled secondary antibody was incubated at room temperature for 1 h, washed, added alkaline phosphatase chromogenic solution to react for 10 min, and the absorption value was detected at 405 nm of the microplate reader, and the positive hole was preliminarily determined that the absorption value was more than 3 times that of the control group. Positive clones were transferred to shake tubes for plasmid extraction and sequencing.
  • Nanobody fragments After preparing the linearized pFUSE-mlgG2b-Fc and pFUSE-hlgG1-Fc vectors, PCR-amplified Nanobody fragments, using homologous recombination to connect Nanobody and vector pFUSE-hlgG1-Fc or pFUSE-mlgG2b-Fc, followed by mammalian cells HEK293F expressed candidate nanobodies and purified them through Protein A affinity column to obtain nanobodies.
  • a preferred Nanobody Nb16 is obtained in the present invention, and the VHH sequence is shown in SEQ ID NO.: 1:
  • the underlined part is the CDR part.
  • nanobodies obtained in the present invention are: Nb9, Nb10, Nb11, Nb17.
  • VHH sequences of Nb9, Nb10, Nb11, Nb17 are shown in SEQ ID NO.: 5, 9, 13, 17 respectively, and the CDR part is shown in Table 1.
  • the plates were coated with mPD-L1-Fc fusion protein at 4°C overnight, and then blocked with BSA at 37°C for 2 hours.
  • Different concentrations of Nb16 nanobody and 10 ⁇ g/mL mouse PD-1-Fc-Biotin fusion protein were added to each well.
  • the reaction was performed at room temperature for 1 hour, and after washing, antibody SA-HRP was added, and the reaction was performed at room temperature for 1 hour. After washing, add color developing solution and read the absorption value at 450nm wavelength.
  • the measurement results show that among the candidate nanobodies in the present invention, some nanobodies (such as Nb9, Nb10, etc.) are candidate antibodies with blocking type, and some nanobodies (such as Nb11, Nb17, etc.) are non-blocking nanobodies.
  • some nanobodies such as Nb9, Nb10, etc.
  • some nanobodies such as Nb11, Nb17, etc.
  • the molecular structure of the TLR7 small molecule agonist used to conjugate the antibody is as follows:
  • SZU-101 and the above-mentioned SZU-101 derivatives for coupling can be carried out by the following methods or similar methods.
  • SZU-101-Mal can be prepared in a similar manner.
  • Example 5 Combined antitumor effect of Nanobody Nb16 and TLR7 agonist SZU-101
  • the mouse CT26 tumor cells were resuscitated and passaged to ensure that the tumor cells had been passed down for at least 3 generations at the time of tumor bearing. Before tumor bearing, the tumor cells were induced with mouse IFN- ⁇ with a final concentration of 100 ng/mL for 24 hours to make the cell surface highly express PD.
  • CT26 tumor cells with high PD-L1 expression after induction were subcutaneously inoculated into female BALB/c mice at a dose of 1 ⁇ 10 6 cells/mice; the mice were randomly divided into 4 groups (9 in each group) only), namely: isotype control group (mFc), nanobody Nb16 group (Nb16), TLR7 agonist SZU-101 group (SZU-101) and combination treatment group (Nb16/SZU-101); among them, mFc and Nb16 were given
  • the dose was 10 mg/kg, intraperitoneal administration; the dose of SZU-101 was 3 mg/kg, peritumoral administration.
  • the dosing cycle was that Nb16 was administered on D1, D5, D9, D12, and D14, and SZU-101 was administered on D9-D14 (3A).
  • Nb16 was administered on D1, D5, D9, D12, and D14
  • SZU-101 was administered on D9-D14 (3A).
  • observe and record the body weight of the mice and the long diameter (L) and short diameter (W) of the tumor until the end of the dissection, calculate the tumor volume V (L ⁇ W ⁇ W)/2, draw the tumor growth curve, and calculate the tumor Inhibition rate; the experiment was terminated on the fifteenth day, and after euthanasia was performed, the animals were dissected and the subcutaneous tumors were removed.
  • the mouse tumor growth curve (3B), end point tumor weight (3C) and end point tumor anatomy photos (3D) are shown in Figure 3.
  • the results showed that both Nb16 and SZU-101 could significantly inhibit tumor growth, and the combination of the two unexpectedly exerted a more significant anti-tumor effect than single drug treatment.
  • the tumor inhibition rates of Nb16 and SZU-101 in the single drug group were 35%, 49%, and the tumor inhibition rate of combined therapy can reach 62%.
  • the end point tumor weight is shown in Table 2.
  • the activated ester was dissolved in DMSO, and the antibody and the small molecule reacted according to the molar ratio of 1:10. A certain amount of the small molecule activated ester was put into Nb16, and the reaction was stirred at 4°C for 4 hours. After the reaction, PBS was added to the mixture to mix, and the small molecules were removed by filtration with a 10kD biological membrane to obtain a novel conjugated compound Nb16-SZU-101.
  • the antibody was first denatured to open the disulfide bond, and then the sample was identified by XevoG2XSQTOF mass spectrometer.
  • the coupling degree of the Nanobody conjugate obtained by mass spectrometry was 4.5.
  • a PD-L1 and TLR7 dual-targeting nanobody conjugated drug was prepared and named Nb16-SZU-101 (Fig. 4A).
  • Example 7 In vitro activity assay of PD-L1 and TLR7 dual-targeted nanobody drug conjugates
  • Example 8 Evaluation of in vivo antitumor activity of PD-L1 and TLR7 dual-targeted nanobody drug conjugates
  • mice were randomly divided into 4 groups: mFc group, Nb16 group, Nb16/SZU-101 combined administration group and Nb16-SZU-101 Nanobody Conjugate Drug Group, wherein the dosage of mFc or Nb16 or Nb16-SZU-101 is 10mg/kg, and the way of administration is intraperitoneal administration, and the dosage of SZU-101 is 0.5mg/kg kg, and the mode of administration was intraperitoneal administration; they were administered on Day 2, Day 6, Day 10, and Day 13, respectively, for a total of 4 doses.
  • Tumor size was measured 2-3 times a week, and tumor growth curves were drawn; and on Day 14, mice were dissected, weighed, and photographed. The tumor growth curve, end-point tumor weight and end-point tumor photos are shown in Figure 7.
  • the experimental results show that the nanobody conjugated drug Nb16-SZU-101 treatment group in the present invention has a significantly improved anti-tumor effect.
  • the tumor inhibition rate of SZU-101 was 39%, and the tumor inhibition rate of the nanobody-conjugated drug group was up to 81%.
  • the end point tumor weight is shown in Table 3. No matter relative to the single-administration group or the combined-administration group, the nanobody-drug conjugate group could significantly inhibit tumor growth.
  • the combined administration group did not significantly inhibit tumor growth, which may be due to the reduced dose of SZU-101 in the combined administration group, and in order to be consistent with the conjugated group, given
  • the drug method was changed to intraperitoneal administration, and intraperitoneal administration was less efficient than peritumoral administration.
  • the CT26 tumor cells before and after induction were subcutaneously inoculated into female BALB/c mice to construct a tumor-bearing mouse model, and the inoculation amount was 1 ⁇ 10 6 cells per mouse; in both models, the mice were randomly divided into two groups. 2 groups, 6 in each group, namely: blank control group mFc (concentration of 10 mg/kg), and conjugated compound Nb16-SZU-101 group (concentration of 10 mg/kg); the administration dose was 200 ⁇ L, and the administration methods were all.
  • mFc concentration of 10 mg/kg
  • Nb16-SZU-101 group concentration of 10 mg/kg
  • the administration dose was 200 ⁇ L, and the administration methods were all.
  • intraperitoneal administration administration on Day 2, Day 5, Day 8, Day 11 respectively, a total of 4 administrations; 2-3 times a week to measure tumor size, draw tumor growth curve; and on Day 12 for mice Anatomical weighing and photographing.
  • the results from the tumor growth curve showed that, compared with the negative control, the conjugated compound could significantly inhibit tumor growth in both tumor models before and after CT26 induction (Fig. 8B), and the inhibition rate in the uninduced CT26 model was 78.9%. %, while the inhibition rate in the CT26 model after induction was 83.6%, and there was no significant difference in the tumor inhibitory effect of the conjugated compound in the two models.
  • the results of the end-point tumor weight (Fig. 8C) and the end-point tumor photo (Fig. 8D) were also basically consistent with the end-point tumor volume results. It was confirmed that the conjugated compound Nb16-SZU-101 exerted a significant inhibitory effect on tumors with low and high expression of PD-L1.
  • the PD-L1 and TLR7 dual-targeted nanobody drug conjugates provided by the present invention not only have significant anti-tumor effects on "hot” tumors with high PD-L1 expression, but also can be used for "cold” tumors with low PD-L1 expression Antitumor therapy of tumors.
  • the present invention further evaluates the PD-L1 and TLR7 dual-targeting nanobody drug conjugates in low-expression PD-L1.
  • mice C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, the mFc group and the nanobody-conjugated drug Nb16-SZU-101 group.
  • 1 ⁇ 10 6 B16-F10 cells that did not induce low PD-L1 expression were seeded subcutaneously in the underarm of mice ( FIG. 9A ).
  • MFc and nanobody conjugated drug Nb16-SZU-101 were used for administration; the control drug and Nb16-SZU-101 were administered at a dose of 10 mg/kg and a volume of 200 ⁇ L; each group was administered by intraperitoneal injection.
  • the mice were euthanized on Day 12.
  • the tumor growth curve, end point tumor weight and end point tumor photos are shown in Figure 9B-D.
  • the nanobody conjugated drug Nb16-SZU-101 treatment group of the present invention has a significantly improved anti-tumor effect.
  • the tumor inhibition rate is about 68.2%, which suggests that the PD-L1 and TLR7 dual-targeted nanobody conjugate drug provided by the present invention can be used for anti-tumor treatment of tumors with low PD-L1 expression.
  • the adopted therapeutic drugs or regimens targeting PD-L1 can usually only target tumors with high PD-L1 expression, and cannot be effectively used for tumors with low or moderate PD-L1 expression. Therefore, it is unexpected that the dual-target nanobody conjugated drug of the present invention can be used to treat tumors with low or moderate PD-L1 expression.
  • uninduced CT26 tumor cells were subcutaneously inoculated into female BALB/c mice to construct a tumor-bearing mouse model, and the inoculation amount was 1 ⁇ 10 6 cells/mice; the mice were divided into 2 models in both models.
  • Groups, 8 in each group namely: blank control group mFc (concentration of 10 mg/kg), and conjugated compound Nb16-SZU-101 group (concentration of 10 mg/kg); the administration dose was 200 ⁇ L, and the administration methods were Intraperitoneal administration.
  • the drug was administered on Day 4, Day 7, Day 10, Day 13, and Day 16 for a total of 5 doses; Day 15, Day 17 administration, a total of 4 administrations. Tumor size was measured 2-3 times a week, and tumor growth curves were drawn.
  • Example 9 Tumor immunophenotyping analysis of PD-L1 and TLR7 dual-targeted nanobody conjugated drug administration
  • the subcutaneous tumor was taken, and 150 mg of tumor tissue was cut into a pulp, digested with hyaluronidase and collagenase IV at 37°C at 180 rpm for 1.5 h, and processed into a single-cell suspension. liquid.
  • the pretreated cell suspension was divided into two parts, one for immunophenotyping analysis of macrophages and dendritic cells, and one for immunophenotyping analysis of T cells and NK cells.
  • cells were treated with 2-4 mL of sterile red blood cell lysate for 8 min and terminated with PBS buffer to remove red blood cells and debris in the sample suspension; cell samples were washed for use.
  • T cell and NK cell sample suspension Treatment of T cell and NK cell sample suspension: The cells are centrifuged with lymphocyte separation solution to obtain the lymphocyte separation layer, which is the tumor lymphocyte suspension, which is washed for later use.
  • Macrophage and dendritic cell samples and T cells and NK cell samples were blocked with Fc receptor blocking solution for 1 hour at 4°C to remove non-specific staining caused by Fc receptors; cell surface protein staining was performed, specific: M1 Macrophages (FITC Rat Anti-Mouse CD45, PE-Cyanine7 Rat Anti-Mouse F4/80 and APC Rat anti-Mouse CD86), M2 macrophages (FITC Rat Anti-Mouse CD45 and PE-Cyanine7 Rat Anti-Mouse F4 /80), dendritic cells (PE-Cy7 Anti-Mouse CD11c, BB515 Rat Anti-Mouse IA/IE, PE Hamster Anti-Mouse CD80 and APC Rat anti-Mouse CD86), CD4+ T cells and CD8+ T cells (FITC Hamster Anti-Mouse CD3e, PerCP-Cy TM 5.5 Rat Anti-Mouse CD4 and APC Rat Anti-Mouse CD8
  • PD-L1 and TLR7 dual-targeting nanobody conjugated drug administration promoted the expression of PD-L1 in macrophages in the intratumoral microenvironment (Fig.
  • Antibody-drug conjugates respond to "cold" tumors with low expression of PD-L1 molecules, achieving a broader response and better anti-tumor efficacy.
  • these immune cells activated by the conjugates of the present invention increase the number of immune cell subtypes that secrete IFN- ⁇ , and IFN- ⁇ is a Induction of PD-L1 expression, one of the most common cytokines, can further promote PD-L1 expression in tumor cells, thereby turning "cold" tumors into “hot” tumors.
  • PD-L1 and TLR7 dual-targeted nanobody drug conjugates target the tumor immune microenvironment, which can reshape the tumor immune microenvironment and coordinate innate and adaptive immunity to exert anti-tumor activity.
  • Example 10 Immune cell deletion analysis to determine the immune basis for the efficacy of PD-L1 and TLR7 dual-targeted nanobody drug conjugates
  • mice aged 6-8 weeks were randomly divided into 6 groups with 6 animals in each group.
  • the corresponding groups are: mFC group, Nb16-SZU-101 group, Nb16-SZU-101+anti-CD4 group, Nb16-SZU-101+anti-CD8 group, Nb16-SZU-101+anti-NK1.1 group, Nb16-SZU-101+chlorophosphoric acid liposome group.
  • the dosing schedule for mFc and Nb16-SZU-101 was the same as in Example 8.1.
  • the immune cell deletion method is: intraperitoneal injection of antibody drugs (anti-CD4, anti-CD8, anti-NK1.1) on Day 3 (200ug/a)/Day 4 (100ug/a)/Day 5 (100ug/a)
  • the corresponding immune cells (CD4+T cells, CD8+T cells, NK cells) were deleted, or macrophages were removed by intraperitoneal injection of chlorophosphoric acid-liposomes.
  • mice were euthanized on Day 14. Tumor growth curves and end-point tumor photographs are shown in Figure 12.
  • the efficacy of PD-L1 and TLR7 dual-targeting nanobody drug conjugates has little effect, which indicates that the in vivo anti-tumor activity of the PD-L1 and TLR7 dual-targeting nanobody drug conjugates provided by the present invention is mainly through CD8+ T cells and NK cells play a role, and killer cells are irreplaceable in drug efficacy.
  • Example 2 As described in Example 1, after expressing the human PD-L1 protein and immunizing camels, a library was constructed and panned, and 11 candidate positive clones were obtained by ELISA identification.
  • the candidate Nanobody sequences were homologously recombined into pFUSE-mlgG2b-Fc and pFUSE-hIgG1-Fc vectors, and then the candidate Nanobody was expressed in mammalian cells HEK293F.
  • 11 preferred anti-human PD-L1 nanobodies are obtained, which are h_Nb1, h_Nb2, h_Nb4, h_Nb5, h_Nb6, h_Nb9, h_Nb12, h_Nb13, h_Nb19, h_Nb26, h_Nb30.
  • VHH sequences of h_Nb1, h_Nb2, h_Nb4, h_Nb5, h_Nb6, h_Nb9, h_Nb12, h_Nb13, h_Nb19, h_Nb26, h_Nb30 are respectively as SEQ ID NO.: 21, 25, 29, 33, 37, 40, 44, 48, 52 , 56, 59, and the CDR part is shown in Table 4.
  • Example 12 Preliminary in vitro activity evaluation of nanobodies against human PD-L1
  • Example 13 In vitro activity evaluation of nanobodies against human PD-L1
  • the stably transfected cell line HEK293T/hPD-L1 was digested to prepare cell samples; different concentrations of nanobody h_Nb1 or h_Nb2 or positive control antibody KN035 were added to the samples; Fc (FITC) was used as a secondary antibody, and incubated for staining; after that, it was detected by flow cytometer to obtain the EC 50 value of the candidate nanobody binding to human PD-L1.
  • Fc FITC
  • the stably transfected cell line HEK293T/hPD-L1 was digested to prepare cell samples; different concentrations of nanobody h_Nb1 or h_Nb2 or positive control antibody KN035, and human PD-1-his protein (concentration) were added to the samples. After incubation and centrifugation, use anti-his-APC as the secondary antibody to incubate for staining; then use flow cytometry to detect on the machine, and obtain candidate nanobodies that block the binding of human PD-1/PD-L1. IC50 value.
  • Example 14 Evaluation of in vivo antitumor activity of anti-human PD-L1 and TLR7 dual-targeted nanobody drug conjugates
  • anti-human PD-L1 and TLR7 dual-targeting nanobody conjugated drugs h_Nb1-SZU-101 and h_Nb2-SZU-101 were prepared and obtained.
  • human antibody FRs were replaced with camel antibody FRs to reduce immunogenicity.
  • homology modeling is carried out on the candidate antibody to identify the key amino acid residues.
  • the candidate nanobody sequence is used as a template to search for the homologous structure in the structure database, and the optimal structural sequence is selected for sequence replacement and finally the human source is obtained.
  • the sequence of the modified antibody should be considered, and the key sites in the framework region that could potentially affect the function of the CDR should be considered.
  • the obtained 11 preferred anti-human PD-L1 nanobodies are respectively humanized, wherein the corresponding sequences of the humanized antibodies are as follows:
  • h_Nb1 The humanized sequences of h_Nb1 are h_Nb1_1, h_Nb1_2, h_Nb1_3, h_Nb1_4, h_Nb1_5;
  • h_Nb2 The humanized sequences of h_Nb2 are h_Nb2_1, h_Nb2_2, h_Nb2_3, h_Nb2_4, h_Nb2_5;
  • h_Nb4 The humanized sequences of h_Nb4 are h_Nb4_1 and h_Nb4_2 respectively;
  • h_Nb5 The humanized sequences of h_Nb5 are h_Nb5_1, h_Nb5_2, h_Nb5_3;
  • h_Nb6_1 and h_Nb6_2 The sequences of h_Nb6 after humanization are h_Nb6_1 and h_Nb6_2 respectively;
  • h_Nb9 The humanized sequences of h_Nb9 are h_Nb9_1 and h_Nb9_2 respectively;
  • h_Nb12_1 and h_Nb12_2 The sequences of h_Nb12 after humanization are h_Nb12_1 and h_Nb12_2 respectively;
  • h_Nb13_1 and h_Nb13_2 The sequences of h_Nb13 after humanization are h_Nb13_1 and h_Nb13_2 respectively;
  • h_Nb19 The humanized sequences of h_Nb19 are h_Nb19_1 and h_Nb19_2 respectively;
  • h_Nb26 The humanized sequences of h_Nb26 are h_Nb26_1 and h_Nb26_2 respectively;
  • h_Nb30 The humanized sequences of h_Nb30 are h_Nb30_1 and h_Nb30_2, respectively.
  • the full-length amino acid sequence (SEQ ID No. 21 and SEQ ID No. 25) of the preferred specific binding human PD-L1 Nanobody were annotated by CCG; Construction, select the appropriate FR region and CDR region template, construct and select the optimal nanobody three-dimensional protein structure; obtain the crystal structure of human PD-L1 protein from the PDB protein database, through protein complex homology modeling and protein-protein Molecular docking method to obtain the preferred PD-L1 nanobody-PD-L1 protein complex structure candidate library; according to the PD-1/PD-L1 in vitro competitive binding characteristics of the preferred antibody, select the appropriate docking angle to determine the optimal docking Conformation; structural analysis of selected nanobody-PD-L1 protein complexes for key interaction sites, focusing on van der Waals forces, hydrogen bonds, ionic bonds, hydrophobic interactions, etc.; focusing on the affinity between nanobodies and PD-L1 acting sites The interaction site pair with lower energy value is used as the target
  • the sequence is SEQ ID No.92-SEQ No.117.
  • the primary antibody corresponding to the sequence shown in SEQ ID NO.92-106 is h_Nb1VHH (SEQ ID NO.21); the primary antibody corresponding to the sequence shown in SEQ ID NO.107-117 is h_Nb2VHH (SEQ ID NO.25 ).
  • PD-L1 antibodies have better targeting and inhibitory effects on highly immunogenic "hot” tumors and tumors with high PD-L1 expression levels, and have better inhibitory effects on tumors with low immunogenicity and low PD-L1 expression poor. Therefore, combination therapy regimens can be based on improving tumor immunogenicity and PD-L1 expression levels to improve treatment efficacy and response rates.
  • a scientifically reasonable, safe and effective combination therapy scheme can also guide the design of new drug molecules.
  • the tumor growth can be significantly inhibited, and a PD-L1 and TLR7 dual-targeted nanobody conjugate was developed.
  • the drug which has achieved efficacy superior to that of combination therapy, can coordinate innate and adaptive immune responses, target and reshape the tumor immune microenvironment, and increase the expression level of PD-L1 in tumor tissue.
  • the tumor model with low expression of PD-L1 also showed extremely significant anti-tumor activity and response rate, showing its clinical application value.
  • the present invention provides for the first time a PD-L1 and TLR7 dual-targeted nanobody conjugated drug, which can be used for subsequent tumor immunotherapy drug development, and is especially suitable for tumors with low immunogenicity and/or low PD-L1 expression.

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Abstract

本发明公开了抗PD-L1纳米抗体和TLR7小分子激动剂的联合治疗抗肿瘤用途，以及PD-L1和TLR7双靶向纳米抗体偶联药物及其制备方法和应用。具体地，本发明公开了联合抗PD-L1的纳米抗体及其衍生蛋白和TLR7小分子激动剂及其衍生化合物用于抗肿瘤治疗的用途和方案，同时本发明公开了新型PD-L1和TLR7双靶向纳米抗体药物偶联物及其衍生分子的设计、制备和鉴定方案及其在抗肿瘤治疗中的作用。本发明的PD-L1和TLR7双靶向纳米抗体药物偶联物可在多种移植瘤模型中发挥显著的抗肿瘤药效。

Description

PD-L1和TLR7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 技术领域
本发明涉及生物医药领域，更具体地涉及一种PD-L1和TLR7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用。
背景技术
肿瘤通过上调表达PD-L1等免疫检查点分子来实现免疫逃逸。针对PD-L1等免疫检查点分子靶点已有多种抗体药物被开发用于***，然而PD-L1抗体等免疫检查点阻断疗法仍面临患者响应率低等问题。联合治疗方案是提高免疫检查点阻断疗法抗肿瘤治疗效果和患者响应率的有效途径。然而，科学合理且安全有效的联合治疗方法仍需深入研究。
Toll样受体(TLR)是固有免疫的重要模式识别受体。TLR受体激动剂可以激活固有免疫反应从而辅助适应性免疫的激活，其中TLR7激动剂被广泛研究并应用于抗肿瘤治疗。TLR激动剂是免疫检查点阻断疗法的潜在联合对象之一。
单克隆抗体在肿瘤免疫治疗中取得了突破性进展。然而，传统单克隆抗体具有分子量大、组织穿透力差和免疫原性高等缺点。而纳米抗体是目前最小的抗体分子，除了具有单克隆抗体的特异性外，具有组织穿透能力强、免疫原性低、稳定性好、人源化简单、易于制备等优势。基于纳米抗体开发的纳米抗体偶联药物是一种新型的药物分子形式，在药物递送、体内成像和抗肿瘤治疗等领域具有广泛应用前景。
综上，本领域急需开发一种新型、有效的PD-L1纳米抗体偶联药物。
发明内容
本发明的目的在于提供一种新型、有效的PD-L1纳米抗体偶联药物。
本发明的目的在于提供一种PD-L1纳米抗体和TLR7激动剂联合抗肿瘤治疗用途以及提供一种PD-L1和TLR7双靶向纳米抗体偶联药物。
本发明的另一目的在于提供PD-L1和TLR7双靶向纳米抗体偶联药物在肿瘤预防和治疗中的应用，特别是PD-L1抗体低响应率的肿瘤中的应用。
在本发明的第一方面，提供了一种抗体-药物偶联物或其药学上可接受的盐，所述的抗体-药物偶联物结构如式Ⅰ所示：
Ab-(J-U)n      (Ⅰ)
式中，
Ab为PD-L1抗体；
U各自独立地为TLR激动剂；
J为化学键或连接子；
n为0或正整数；
“-”为化学键或接头或连接子。
在另一优选例中，所述的PD-L1抗体包括单特异性抗体、双特异性抗体、多特异性抗体(如三特异性抗体)。
在另一优选例中，所述的PD-L1抗体包括：单克隆抗体、单链抗体(scFv)、纳米抗体。
在另一优选例中，所述的PD-L1抗体包括单价、二价或多价抗体。
在另一优选例中，所述的PD-L1抗体包括多聚体形式的抗体。
在另一优选例中，所述的PD-L1抗体特异性结合PD-L1。
在另一优选例中，所述的PD-L1抗体包括PD-L1单价纳米抗体、二价纳米抗体和/或多价纳米抗体。
在另一优选例中，所述的PD-L1抗体包括阻断型(可阻断PD-L1和PD-1的结合)、非阻断型(不阻断PD-L1和PD-1的结合)、或其组合。
在另一优选例中，所述的PD-L1抗体为阻断型抗体。
在另一优选例中，所述的PD-L1抗体阻断PD-1与PD-L1的结合。
在另一优选例中，所述的PD-L1为人PD-L1或非人哺乳动物的PD-L1(如小鼠PD-L1)。
在另一优选例中，所述的PD-L1抗体为人或非人哺乳动物抗体。
在另一优选例中，所述非人哺乳动物选自下组：骆驼、羊驼、小鼠、食蟹猴。
在另一优选例中，所述的PD-L1抗体为PD-L1纳米抗体或其衍生抗体。
在另一优选例中，所述的衍生抗体为针对PD-L1纳米抗体的修饰改造，包括但不限于将PD-L1纳米抗体连接Fc片段、人血清白蛋白、聚乙二醇PEG、形成二价抗体和/或多价抗体。
在另一优选例中，所述的纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在另一优选例中，所述的PD-L1纳米抗体特异性结合PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
(1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
(2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
(4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；和
(5)SEQ ID NO：18所示的CDR1、SEQ ID NO：19所示的CDR2，SEQ ID NO：20所示的CDR3。
在另一优选例中，所述的PD-L1纳米抗体特异性结合人PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
(6)SEQ ID NO：22所示的CDR1、SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3；
(7)SEQ ID NO：26所示的CDR1、SEQ ID NO：27所示的CDR2，SEQ ID NO：28所示的CDR3；
(8)SEQ ID NO：30所示的CDR1、SEQ ID NO：31所示的CDR2，SEQ ID NO：32所示的CDR3；
(9)SEQ ID NO：34所示的CDR1、SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3；
(10)SEQ ID NO：22所示的CDR1、SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3；
(11)SEQ ID NO：41所示的CDR1、SEQ ID NO：42所示的CDR2，SEQ ID NO：43所示的CDR3；
(12)SEQ ID NO：45所示的CDR1、SEQ ID NO：46所示的CDR2，SEQ ID NO：47所示的CDR3；
(13)SEQ ID NO：49所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3；
(14)SEQ ID NO：53所示的CDR1、SEQ ID NO：54所示的CDR2，SEQ ID NO：55所示的CDR3；
(15)SEQ ID NO：57所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：58所示的CDR3；和
(16)SEQ ID NO：60所示的CDR1、SEQ ID NO：61所示的CDR2，SEQ ID NO：62所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍生序列。
在另一优选例中，所述抗PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、或SEQ ID NO:17中所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:1、SEQ ID NO:5、SEQ ID  NO:9、SEQ ID NO:13、或SEQ ID NO:17具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括特异性结合人PD-L1纳米抗体。
在另一优选例中，所述特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:21、25、29、33、37、40、44、48、52、56、59所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:21、25、29、33、37、40、44、48、52、56或59具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括人源化的特异性结合人PD-L1纳米抗体。
在另一优选例中，所述人源化的特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90或91具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括亲和力成熟的特异性结合人PD-L1纳米抗体。
在另一优选例中，所述亲和力成熟的特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116或117所示的氨基酸 序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述“亲和力成熟”是指，亲和力成熟化改造的抗人PD-L1纳米抗体对PD-L1的亲和力相对于改造前的抗人PD-L1纳米抗体对PD-L1的亲和力，提高了至少1倍、至少2倍、至少3倍、至少4倍、至少5倍、至少6倍、至少7倍、至少8倍、至少9倍、至少10倍、至少11倍、至少12倍、至少20倍、或至少25倍。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116或117具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述纳米抗体的VHH链的互补决定区CDR由SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3组成。
在另一优选例中，所述纳米抗体的VHH链序列如SEQ ID NO.:1所示。在另一优选例中，所述TLR激动剂为大分子(蛋白质或核酸)或小分子激动剂。
在另一优选例中，所述TLR激动剂包括但不限于TLR1激动剂、TLR2激动剂、TLR3激动剂、TLR4激动剂、TLR5激动剂、TLR6激动剂、TLR7激动剂、TLR8激动剂和TLR9激动剂。
在另一优选例中，n为所述抗体-药物偶联物中的药物平均偶联数量，较佳地n为1～9，优选为2.5～6.5，更优选为3.5～5.5。
在另一优选例中，所述的TLR激动剂为TLR7激动剂。
在另一优选例中，所述的TLR激动剂不具有TLR8激动活性。
在另一优选例中，所述的TLR7激动剂为宿主内源性激动剂或外源性激动剂。
在另一优选例中，所述的TLR7激动剂为小分子激动剂。
在另一优选例中，所述的TLR7激动剂包括：SZU-101：
Figure PCTCN2022080953-appb-000001
在另一优选例中，所述TLR7激动剂为SZU-101的衍生化合物，包括但不限于在SZU-101基础进行的一个或多个基团的替换、修饰或删除。
在另一优选例中，所述TLR7激动剂为SZU-101的多价化合物。
在另一优选例中，所述TLR7激动剂(如SZU-101)连接于PD-L1抗体的重链恒定区 或重链可变结构域(VHH)的末端氨基或侧链氨基。
在另一优选例中，所述TLR7激动剂(如SZU-101)连接于PD-L1抗体的巯基。
在另一优选例中，所述SZU-101连接于PD-L1抗体的氨基，并形成S1所示结构：
Figure PCTCN2022080953-appb-000002
或者
所述的SZU-101连接于PD-L1抗体的巯基，并形成S2所示结构：
Figure PCTCN2022080953-appb-000003
在另一优选例中，所述的TLR7激动剂定点和/或随机地连接于所述的PD-L1抗体(即式I中，所述的U定点和/或随机地连接于Z)。
在另一优选例中，所述的U定点连接于Z。
在另一优选例中，所述的U定点连接于PD-L1抗体Z的选自下组的氨基酸位点：G、K、L、A、C或其组合。
在另一优选例中，所述化学键为聚乙二醇PEG。
在另一优选例中，所述化学键为PEG的衍生化合物，包括但不限于在SZU-101的基础上进行的一个或多个基团的替换、修饰或删除。
在另一优选例中，所述PEG化学键的聚合度为大于等于1的正整数。
在另一优选例中，所述抗体-药物偶联物提高肿瘤内细胞的PD-L1水平。
在另一优选例中，所述抗体-药物偶联物激活免疫细胞。
在另一优选例中，所述的激活为体外激活。
在另一优选例中，所述的体外激活包括：在所述的抗体-药物偶联物存在下，培养所述的免疫细胞一段时间(如6-48小时)，从而获得经免疫激活的免疫细胞。
在另一优选例中，所述的免疫细胞选自但不限于：CD8+T细胞、自然杀伤细胞NK、树突状细胞、淋巴细胞、单核/巨噬细胞、粒细胞、或其组合。
在另一优选例中，所述的抗体-药物偶联物或其药学上可接受的盐用于制备一种组合物或制剂，所述组合物或制剂用于：
(a)促进树突状细胞的成熟；
(b)增加肿瘤浸润细胞毒性细胞(CD8+T细胞和NK细胞)的功能；
(c)促进肿瘤浸润细胞毒性细胞颗粒酶B和IFN-γ的表达；
(d)促使肿瘤相关巨噬细胞重极化；
(e)减少TGF-β+巨噬细胞的浸润；
(f)促进IFN-γ+CD4+T细胞的浸润；
(g)促进瘤内巨噬细胞表达PD-L1；
(h)靶向并重塑肿瘤免疫微环境；
(i)提高肿瘤细胞的PD-L1水平；和/或
(j)用于治疗中表达或低表达PD-L1的肿瘤。
在另一优选例中，所述的重塑肿瘤免疫微环境为协调肿瘤内先天免疫和适应性免疫抗肿瘤免疫应答。
在另一优选例中，所述的重塑肿瘤免疫微环境为提高抗肿瘤免疫细胞的浸润，和降低免疫抑制细胞的比例。
在另一优选例中，所述的抗肿瘤免疫细胞包括但不限于分泌颗粒酶及IFN-γ的CD8+T细胞和NK细胞、活化的树突状细胞、分泌IFN-γ的CD4+T细胞、M1巨噬细胞。
在另一优选例中，所述的免疫抑制细胞包括但不限于M2巨噬细胞、Treg细胞、分泌TGF-β的白细胞。
在另一优选例中，所述的PD-L1水平包括细胞表面PD-L1水平和细胞内PD-L1水平。
在另一优选例中，所述的低表达PD-L1的肿瘤为实体瘤或血液瘤。
在本发明的第二方面，提供了一种药物组合物，所述药物组合物包含：
(a)如本发明第一方面所述的抗体-药物偶联物或其药学上可接受的盐；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物还包含：
(c)其他生物活性的药物，如***的药物。
在另一优选例中，所述其他生物活性的药物促进CD8+T细胞和NK细胞的抗肿瘤功能。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物的施用方式选自下组：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述药物组合物的施用方式为，将所述药物组合物和免疫细胞(如树突状细胞、自然杀伤细胞、淋巴细胞、单核/巨噬细胞、粒细胞等)共培养后，分离免疫细胞进行体内回输。
在另一优选例中，所述的药物组合物的剂型选自下组：液态、固体、或凝胶态。
在另一优选例中，所述的药物组合物用于抗肿瘤治疗。
在另一优选例中，所述的药物组合物用于治疗PD-L1低表达的肿瘤。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1低于正常肿瘤表达PD-L1的量E0，较佳地E1/E0≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
在本发明的第三方面，提供了一种免疫偶联物，所述的免疫偶联物含有：
(a)如本发明第一方面所述的抗体-药物偶联物；和
(b)其他偶联部分。
在另一优选例中，所述其他的偶联部分选自下组：小分子化合物、PEG、荧光素、放射性同位素、造影剂、脂肪酸链、蛋白片段、或其组合。
在另一优选例中，所述的组分(a)和(b)可操作性连接。
在另一优选例中，所述的偶联部分包括化学标记和生物标记。
在另一优选例中，所述化学标记选自同位素、免疫毒素和/或化学药物。
在另一优选例中，所述生物标记选自生物素、亲和素或酶标记。
在另一优选例中，所述小分子化合物选自***或自身免疫性疾病药物或毒素。
在另一优选例中，所述的放射性同位素包括：
(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-133、Yb-169、Yb-177，或其组合。
在另一优选例中，所述的放射性同位素包括但不限于碘131、铟111和镥177。
在另一优选例中，所述的造影剂用于MRI或CT。
在另一优选例中，所述的蛋白片段包括但不限于抗体Fc、生物素、亲和素、HRP、抗体、酶、细胞因子及其他生物活性蛋白或多肽。
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，所述偶联部分选自下组：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等)、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(例如， DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在本发明的第四方面，提供了一种融合蛋白，所述的融合蛋白包含：
(a)如本发明第八方面所述的PD-L1纳米抗体；和
(b)任选的具有治疗功能的多肽分子和蛋白片段。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括但不限于：靶向PD-1、IL-4R、IL-4Rα、TNF-α、VEGF、4-1BB、CD47、TIM3、CTLA4、IL-17A、CD19、CD22、CD28、CD38、CD40、CD47、B7-H3、TSLP、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、HER2的多肽分子或片段。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括但不限于：胰岛素、IL-2、干扰素、降钙素、GHRH肽、肠肽类似物、白蛋白、抗体片段、细胞因子。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括单链抗体(scFv)、双链抗体、单克隆抗体、或嵌合抗体。
在另一优选例中，所述融合蛋白还包含协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列选自下组：6His标签、GGGS序列、FLAG标签。
在另一优选例中，所述的融合蛋白包括双特异性抗体、嵌合抗体。
在本发明的第五方面，提供了一种多特异性抗体，所述的多特异性抗体包含：
(a)如本发明第八方面所述的PD-L1纳米抗体；和
(b)任选的靶向第二抗原的抗体分子。
在另一优选例中，多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：PD-1、IL-4R、IL-4Rα、TNF-α、VEGF、4-1BB、CD47、TIM3、CTLA4、IL-17A、CD19、CD22、CD28、CD38、CD40、CD47、B7-H3、TSLP、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、HER2或其组合。
在另一优选例中，所述的第二抗原结合区为纳米抗体。
在另一优选例中，所述多特异性抗体包括一个或多个第二抗原结合区。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
在本发明的第六方面，提供了一种制备本发明第一方面所述的抗体-药物偶联物的方法，所述方法包括步骤：
配置反应体系，所述反应体系中包括抗体和游离的药物分子，然后进行偶联反应，从而制得所述抗体-药物偶联物，其中，所述药物分子包括TLR激动剂、接头。
在另一优选例中，反应时间为3h-10h。
在另一优选例中，所述抗体与药物分子的摩尔比为1-2：3-20；优选为1：6-10。
在另一优选例中，将SZU-101、EDCI和NHS溶于DMSO中室温搅拌三小时反应制备SZU-101-NHS活性酯，PD-L1纳米抗体和SZU-101-NHS活性酯按照1:10的摩尔比剂量4℃搅拌反应4小时，制备获得纳米抗体偶联药物。
在本发明的第七方面，提供了一种预防或***的方法，向有需要的受试者施用如本发明第一方面所述的纳米抗体偶联药物。
在另一优选例中，所述的肿瘤为表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤选自下组：高表达PD-L1的肿瘤、中表达PD-L1的肿瘤、低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为中表达PD-L1的肿瘤或低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为低表达PD-L1的肿瘤。
在另一优选例中，“高表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)＞1，更佳地≥1.5，更佳地≥2.0。
在另一优选例中，“中表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)为0.5-1.1，更佳地为0.7-1.0，更佳地为0.8-0.9。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
在本发明的第八方面，提供了一种PD-L1纳米抗体，所述PD-L1纳米抗体特异性结合PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
(1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
(2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
(4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；和
(5)SEQ ID NO：18所示的CDR1、SEQ ID NO：19所示的CDR2，SEQ ID NO：20所示的CDR3。
在另一优选例中，所述的PD-L1纳米抗体特异性结合PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
(6)SEQ ID NO：22所示的CDR1、SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3；
(7)SEQ ID NO：26所示的CDR1、SEQ ID NO：27所示的CDR2，SEQ ID NO：28所示的CDR3；
(8)SEQ ID NO：30所示的CDR1、SEQ ID NO：31所示的CDR2，SEQ ID NO：32所示的CDR3；
(9)SEQ ID NO：34所示的CDR1、SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3；
(10)SEQ ID NO：22所示的CDR1、SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3；
(11)SEQ ID NO：41所示的CDR1、SEQ ID NO：42所示的CDR2，SEQ ID NO：43所示的CDR3；
(12)SEQ ID NO：45所示的CDR1、SEQ ID NO：46所示的CDR2，SEQ ID NO：47所示的CDR3；
(13)SEQ ID NO：49所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3；
(14)SEQ ID NO：53所示的CDR1、SEQ ID NO：54所示的CDR2，SEQ ID NO：55所示的CDR3；
(15)SEQ ID NO：57所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：58所示的CDR3；和
(16)SEQ ID NO：60所示的CDR1、SEQ ID NO：61所示的CDR2，SEQ ID NO：62所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍生序列。
在另一优选例中，所述抗PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、或SEQ ID NO:17中所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、或SEQ ID NO:17具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括特异性结合人PD-L1纳米抗体。
在另一优选例中，所述特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:21、25、29、33、37、40、44、48、52、56、59所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:21、25、29、33、37、40、44、48、52、56或59具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括人源化的特异性结合人PD-L1纳米抗体。
在另一优选例中，所述人源化的特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90或91具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体还包括亲和力成熟的特异性结合人PD-L1纳米抗体。
在另一优选例中，所述亲和力成熟的特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116或117所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或 1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116或117具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述“亲和力成熟”是指，亲和力成熟化改造的抗人PD-L1纳米抗体对PD-L1的亲和力相对于改造前的抗人PD-L1纳米抗体对PD-L1的亲和力，提高了至少1倍、至少2倍、至少3倍、至少4倍、至少5倍、至少6倍、至少7倍、至少8倍、至少9倍、至少10倍、至少11倍、至少12倍、至少20倍、或至少25倍。
在本发明的第九方面，提供了一种药盒，所述的药盒包括：
(1)第一容器，以及位于所述第一容器内的如本发明的第八方面所述的PD-L1纳米抗体，以及药学上可用的载体；
(2)第二容器，以及位于所述第二容器内的TLR7激动剂，以及药学上可用的载体；
以及(3)任选的使用说明书。
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ELISA测定纳米抗体Nb16与PD-L1结合的EC50曲线。
图2显示了ELISA测定纳米抗体Nb16阻断PD-1与PD-L1结合的IC50曲线。
图3显示了PD-L1纳米抗体和TLR7激动剂联合治疗的体内抗肿瘤作用，其中，3A为实验荷瘤、给药天数示意图；3B为小鼠肿瘤生长曲线；3C为终点瘤重；3D为终点肿瘤解剖照片。
图4显示了PD-L1和TLR7双靶向纳米抗体偶联药物的结构示意(4A)和偶联度前后的质谱鉴定(4B和4C)。
图5显示了流式细胞术测定PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101与PD-L1结合的EC50曲线，其中纳米抗体Nb16为对照。
图6显示了流式细胞术测定PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101阻断PD-1与PD-L1结合的IC50曲线，其中纳米抗体Nb16为对照。
图7显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101在诱导的CT26肿瘤模型中的抗肿瘤作用，其中7A为小鼠肿瘤生长曲线；7B为终点瘤重；7C为终点肿瘤解剖照片。
图8显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101在未诱导的以及诱导的CT26肿瘤模型中的抗肿瘤作用，其中8A为IFN-γ诱导(PD-L1高表达)和未用IFN-γ诱导(PD-L1低表达)的CT26细胞表面PD-L1表达量的比对图；8B为小鼠肿瘤生长曲线；8C为终点瘤重；8D为终点肿瘤解剖照片。
图9显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101在B16肿瘤模型中的抗肿瘤作用，其中9A为未诱导低表达PD-L1的B16-F10细胞的PD-L1表达示意图，blank为空白对照组，isotype control为同种型对照组；9B为小鼠肿瘤生长曲线；9C为终点瘤重；9D为终点肿瘤解剖照片。
图10显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101在CT26早期模型(肿瘤体积<50mm 3)以及晚期模型(肿瘤体积>200mm 3)中的肿瘤抑制作用，其中10A为早期模型中的小鼠肿瘤生长曲线，10B为早期模型中的小鼠肿瘤生存曲线；10C为晚期模型中的小鼠肿瘤生长曲线，10D为晚期模型中的小鼠肿瘤生存曲线；10E为再荷瘤模型中小鼠的肿瘤生长曲线。
图11显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101给药重塑肿瘤免疫微环境，其中，total为总细胞量，mFc为同型对照组(mFc)，Nb16-SZU-101为纳米抗体偶联药物组。
图12显示了PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101给药时通过CD8+T细胞和NK细胞发挥抗肿瘤作用。其中，12A为肿瘤生长曲线；12B为终点瘤重；12C为终点肿瘤照片。
图13显示了候选抗人PD-L1的纳米抗体的human PD-L1结合活性，其中blank为阴性对照。
图14显示了候选抗人PD-L1的纳米抗体对human PD-1/PD-L1结合的阻断活性测定，其中blank和blank+ligand为对照组。
具体实施方式
发明人通过广泛而深入的研究，首次筛选并鉴定了一种PD-L1纳米抗体，并开发了一种PD-L1和TLR7双靶向纳米抗体偶联药物。具体地，在多种小鼠移植瘤模型中发现，本发明的双靶向纳米抗体偶联药物具有优异的抗肿瘤活性。此外，本发明还出乎意料地发现，本发明的双靶向纳米抗体偶联药物促进瘤内巨噬细胞表达PD-L1，并主要通过CD8+T细胞和NK细胞发挥体内抗肿瘤活性，有利于治疗低表达PD-L1分子的“冷”肿瘤。本发明开发的PD-L1和TLR7双靶向纳米抗体偶联药物表现出突出的抗肿瘤效果和新颖的作用机制，具备临床开发和应用价值。在此基础上完成了本发明。
TLR受体及TLR受体激动剂
如本文所用，术语“TLR受体”是指Toll样受体，是生物体免疫***中一类重要 的固有免疫模式识别受体，可特异性的识别病原微生物进化过程中相对保守的抗原分子(或称为病原相关分子模式)，实现病原微生物入侵的有效检测和固有免疫应答诱导。在人体中已发现十种TLR受体，即TLR1-TLR10,其中TLR3、TLR7、TLR8、TLR9定位于细胞的内体、溶酶体膜上，其余定位于细胞质膜上。在本发明实施方式中，优选TLR7作为药物分子靶标之一。TLR7分子的天然配体为单链线性RNA。
如本文所用，术语“TLR受体激动剂”是指可以特异性结合并激活TLR受体的大分子(蛋白质或核酸)或小分子激动剂，促进TLR受体的下游信号的转导，实现固有免疫细胞的活化。在本发明实施方式中，优选TLR7激动剂构建纳米抗体偶联药物。除本发明实施方式中应用的SZU-101外，可用的TLR7激动剂还包括咪喹莫特和R848等。
如本文所用，术语“本发明纳米抗体”、“本发明的靶向PD-L1的纳米抗体”、“本发明的抗PD-L1纳米抗体”可互换使用，均指特异性识别和结合于PD-L1(包括人或鼠PD-L1)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO:1所示的纳米抗体(Nb16)。
如本文所用，术语“本发明纳米抗体偶联药物”、“本发明的双靶向纳米抗体偶联药物”、“本发明的PD-L1和TLR7双靶向纳米抗体偶联药物”，可互换使用，均指特异性识别和结合于PD-L1(包括人或鼠PD-L1)的纳米抗体及其衍生蛋白偶联TLR7激动剂形成的新型药物分子。纳米抗体偶联药物中的纳米抗体特别优选的是VHH链的氨基酸序列如SEQ ID NO:1所示的纳米抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体(single domain antibody，sdAb，或VHH)”、“纳米抗体”(nanobody)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，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)。
纳米抗体/单域抗体(Nanobody)作为一种新型的小分子抗体片段，由驼类天然的重链抗体重链可变区(VHH)克隆获得。Nanobody(Nb)具有优良的生物学特性，分子量12-15kDa，是完整抗体的十分之一，具有很好的组织穿透性，特异性高，水溶性好。因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，逐渐成为新一代抗体治疗中的 新兴力量，在免疫诊断和治疗中显示出广阔的应用前景。
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β-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β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。
如本文所用，术语“纳米抗体偶联药物”与“纳米抗体药物偶联物”可互换使用。如本领域技术人员所知，纳米抗体偶联物是一种特殊的抗体药物偶联药物形式，其是将纳米抗体或者衍生蛋白偶联药物、毒素、细胞因子、放射性核素、酶和其他诊断或治疗分子形成的药物分子形式，可用于肿瘤治疗、药物递送和体内成像等，具有广阔的临床应用价值。
如本文所用，术语“重链可变区”与“V H”可互换使用。
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结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述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，如包含纳米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纳米抗体的片段。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.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.疗剂(例如，顺铂)或任何形式的纳米颗粒等。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体-药物偶联物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物或具有促进免疫功能的药物。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二 酰亚胺连接到抗体上的马来酰亚胺。
应理解，药物通常可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在本发明中，药物为激活或促进免疫反应的药物，例如激活固有免疫反应从而辅助适应性免疫的激活。在具体的实施方式中，药物为TLR受体激动剂。
在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，TLR1激动剂、TLR2激动剂、TLR3激动剂、TLR4激动剂、TLR5激动剂、TLR6激动剂、TLR7激动剂、TLR8激动剂和TLR9激动剂，例如SZU-101、咪喹莫特、R848、CpG等。在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Ab-(J-U)n      (Ⅰ)
式中，
Ab为PD-L1抗体；
U各自独立地为TLR激动剂；
J为化学键或连接子；
n为0或正整数；
“-”为化学键或接头或连接子。
应用
本发明提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗PD-L1相关疾病的药物。所述PD-L1相关疾病包括炎症疾病、自身免疫疾病等，包括但不限于乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、***癌、结直肠癌、***癌、***、淋巴癌、骨癌、肾上腺肿瘤、或***。
应理解，对一种或多种检查点抑制剂(例如结合PD-L1、CTLA-4或CD47等的抗体)的治疗无反应的癌症，例如胰腺癌或***癌，这种肿瘤被称为冷肿瘤。对用一种或多种检查点抑制剂，例如结合PD-L1、CTLA-4或CD47等的抗体的治疗有反应的癌症，此类肿瘤也称为温或热肿瘤。与不响应检查点抑制剂治疗的肿瘤相比，此类肿瘤被认为具有更高的肿瘤浸润淋巴细胞(TIL)水平和/或更高的肿瘤突变负荷。
本发明提供的一种优选的抗体-药物偶联物，在冷肿瘤和低表达PD-L1的肿瘤模型中同样表现出极其显著的抗肿瘤活性和响应率，在多种移植瘤模型中发挥显著的抗肿瘤药效。
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或赋形剂，以及任选的其他生物活性物质。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)：腹膜内、静脉内、或局部给药。
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。
本发明的主要优点包括：
(a)本发明首次提供了PD-L1纳米抗体和TLR7激动剂的联合治疗方案，表现出了显著的体内抗肿瘤活性，表明PD-L1抗体疗法和TLR7免疫激动剂具有联合合理性，可协同抗肿瘤。
(b)本发明首次开发了PD-L1和TLR7双靶向纳米抗体偶联药物，其可以促进肿瘤中细胞上调表达PD-L1，并协调瘤内先天免疫和适应性抗肿瘤免疫应答，使得其在“冷”肿瘤和低PD-L1表达的肿瘤等PD-L1抗体治疗效果较差的多种肿瘤中表现出极佳的抑制肿瘤生长作用。
(c)本发明首次提供的PD-L1和TLR7双靶向纳米抗体偶联药物可靶向肿瘤免疫微环境，并重塑肿瘤免疫微环境，提高抗肿瘤免疫细胞的浸润，减少免疫抑制细胞的浸润。
(d)本发明首次提供的PD-L1和TLR7双靶向纳米抗体偶联药物发挥抗肿瘤药效机制明确，主要依赖于CD8+T细胞和NK细胞发挥肿瘤杀伤和抑制作用。
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。
实施例1：抗PD-L1的纳米抗体筛选
1.1 PD-L1蛋白表达
构建mouse PD-L1(ECD)-pFUSE-hIgG1-Fc载体，转化进DH5α化学感受态细胞里，利用含有博来霉素抗性的LB固体培养基平板筛选获得单克隆菌株，接菌提取质粒。利用HEK293F细胞哺乳动物表达鼠的PD-L1蛋白，使用无血清培养基对HEK293F细胞进行培养，基于PEI的质粒络合，完成转染，表达蛋白5天后收集蛋白上清，通过Protein A亲和柱纯化蛋白。
1.2免疫动物和文库构建
(1)选取成年新疆骆驼，共需7次免疫鼠的PD-L1蛋白，首次免疫采用弗式完全佐剂，后续加强免疫采用弗式不完全佐剂；(2)免疫结束后3-5天内，从骆驼身上抽取外周血，分离外周血淋巴细胞，Trizol法提取RNA，反转录得到cDNA；(3)通过两步巢式PCR扩增获得VHH基因片段；(4)通过限制性酶PstI和NotI酶切将VHH基因片段连接到pMECS载体上；(5)将连接产物电转化至大肠杆菌电转感受态TG1中，构建鼠PD-L1 蛋白的单域抗体文库；(6)对文库进行检定，通过稀释涂板，计算得到文库的库容大小为5.2×10 9CFU；利用菌落PCR随机选择24个菌落检测文库***率，得到***率为100％。以上结果表明一个高质量的靶向鼠PD-L1的特异性纳米抗体文库构建成功。
1.3文库淘选
向10倍库容的纳米抗体库中加入辅助噬菌体扩增得到纳米抗体展示噬菌体。96孔酶标板用5μg/mL NeutrAvidin溶液(每孔100μL)包被，4℃,包被过夜。第二天，用2％脱脂奶粉室温封闭2h，用20mM HEPES(pH7.5),150mM NaCl溶液洗5次。设置对照组和实验组，加入100μL 200nM PD-L1-biotin蛋白稀释液，室温振荡(700rpm)并孵育15min，用20mM HEPES(pH7.5),150mM NaCl溶液洗5次。之后加入100μL噬菌体稀释液(1×10 13cfu/mL)，室温振荡(700rpm)并孵育2h，用20mM HEPES(pH7.5),150mM NaCl溶液洗涤5次除去不结合的噬菌体。后加入100μL0.25mg/mL的胰酶室温振荡(700rpm)消化30min将特异性结合的噬菌体解离下，加入抑制剂终止消化。将洗脱的噬菌体感染TG1细胞，以便第二轮panning使用。重复上述操作2-3轮，直至阳性克隆被富集。
1.5 ELISA鉴定阳性克隆
几轮淘选过后，将洗脱下来的噬菌体感染处于生长对数期的TG1感受态，梯度稀释并涂布在平板上培养过夜。分别挑取96个克隆接种到每孔100μL培养基的96孔圆底板中静置过夜作为母板，之后吸取10μL过夜培养的菌液到每孔1mL培养基的96孔深底板中，诱导纳米抗体表达并粗纯。96孔酶标板用5μg/mL NeutrAvidin溶液包被，4℃,700rpm,过夜。第二天，加入2％脱脂奶粉室温封闭板子，用含牛血清白蛋白BSA的溶液洗3次。加入100μL 3μg/mL PD-L1蛋白室温孵育30分钟后洗涤，加入粗提的纳米抗体室温孵育1h后洗涤，加入小鼠抗HA一抗室温孵育1小时后洗涤，加入山羊抗小鼠碱性磷酸酶标记二抗室温孵育1h后洗涤，加入碱性磷酸酶显色液反应10min，在酶标仪405nm处检测吸收值，初步判定吸收值是对照组吸收值的3倍以上的即为阳性孔，将阳性克隆转移到摇菌管中培养以便提取质粒并进行测序。
实施例2：抗PD-L1纳米抗体的表达和鉴定
制备线性化pFUSE-mIgG2b-Fc和pFUSE-hIgG1-Fc载体后，PCR扩增纳米抗体片段，利用同源重组连接纳米抗体和载体pFUSE-hIgG1-Fc或pFUSE-mIgG2b-Fc，随后利用哺乳动物细胞HEK293F表达候选纳米抗体，并通过Protein A亲和柱纯化获得纳米抗体。
本发明中获得一优选的纳米抗体Nb16，VHH序列如SEQ ID NO.:1所示：
Figure PCTCN2022080953-appb-000004
其中，下划线标示的为CDR部分。
此外，本发明中获得其它几株纳米抗体，分别为：Nb9、Nb10、Nb11、Nb17。
其中，Nb9、Nb10、Nb11、Nb17的VHH序列分别如SEQ ID NO.:5、9、13、17所示，CDR部分见表1。
表1.抗体的VHH及CDR序列
Figure PCTCN2022080953-appb-000005
Figure PCTCN2022080953-appb-000006
实施例3：抗PD-L1纳米抗体的体外活性评价
3.1候选纳米抗体Nb16的PD-L1结合活性测定
用mPD-L1-Fc融合蛋白包被平板4℃过夜，之后用BSA于37℃封闭2小时，每孔加入不同浓度的Nb16纳米抗体，室温下反应1小时，洗涤后加入山羊抗小鼠辣根过氧化物酶标记抗体，室温反应1小时。洗涤后加入显色液，450nm波长读取吸收值。
实验结果表明，该反应中，纳米抗体Nb16的EC50曲线见图1，为0.045μg/mL。
3.2候选纳米抗体Nb16的PD-1/PD-L1阻断活性测定
用mPD-L1-Fc融合蛋白包被平板4℃过夜，之后用BSA在37℃封闭2小时，每孔加入不同浓度的Nb16纳米抗体以及10μg/mL小鼠PD-1-Fc-Biotin融合蛋白，室温下反应1小时，洗涤后加入抗体SA-HRP，室温反应1小时。洗涤后加入显色液，450nm波长读取吸收值。
实验结果表明，该反应中，纳米抗体Nb16的IC 50曲线见图2，为1.017μg/mL。可以有效阻断PD-1/PD-L1的结合。
3.3候选纳米抗体Nb9、Nb10、Nb11、Nb17的体外活性
测定结果表明，本发明中候选纳米抗体中，部分纳米抗体(如Nb9、Nb10等)为具有阻断型的候选抗体，部分纳米抗体(如Nb11、Nb17等)为非阻断型纳米抗体。
实施例4：TLR7激动剂SZU-101的制备
TLR7小分子激动剂用于偶联抗体的分子结构如下：
Figure PCTCN2022080953-appb-000007
SZU-101及上述用于偶联的SZU-101衍生物的合成可采用以下方法或类似方法。
Figure PCTCN2022080953-appb-000008
将化合物20-1溶解于无水DMSO中，于10℃冷却加入等当量的丁二酸酐，混合物自然室温搅拌24小时。混合反应物倒与20倍体积水中，析出大量白色固体化合物SZU-101。
将SZU-101(1eq),NHS(1.2eq)和EDC(1.3eq)溶于无水DMF，室温下搅拌4h，结束反应，将反应液倒入二氯甲烷中，抽滤干燥，得化合物23，即SZU-101-NHS，为白色固体。
SZU-101-Mal可用类似方法制备。
实施例5：纳米抗体Nb16和TLR7激动剂SZU-101的联合抗肿瘤作用
复苏小鼠CT26肿瘤细胞并传代，保证荷瘤时肿瘤细胞至少已经传了3代，荷瘤前用终浓度为100ng/mL的小鼠IFN-γ诱导肿瘤细胞24h，使其细胞表面高表达PD-L1；将诱导后高表达PD-L1的CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中，接种量为1×10 6细胞/只；并将小鼠随机分组为4组(每组9只)，即：同型对照组(mFc)、纳米抗体Nb16组(Nb16)、TLR7激动剂SZU-101组(SZU-101)和联合治疗组(Nb16/SZU-101)；其中，mFc和Nb16给药量为10mg/kg，腹腔给药；SZU-101给药量为3mg/kg，瘤周给药。给药周期为Nb16于D1、D5、D9、D12、D14给药，SZU-101于D9-D14给药(3A)。给药周期中观察并记录小鼠体重和肿瘤长径(L)与短径(W)，直至解剖终点，计算瘤体积V＝(L×W×W)/2，绘制肿瘤生长曲线，计算肿瘤抑制率；第十五天结束实验，执行安乐死后，解剖动物，取出皮下肿瘤。
小鼠肿瘤生长曲线(3B)、终点瘤重(3C)和终点肿瘤解剖照片(3D)如图3所示。结果表明，Nb16和SZU-101均可以显著抑制肿瘤生长，而二者联用意外地发挥比单药治疗更显著的抗肿瘤作用，其中：单独给药组Nb16和SZU-101的抑瘤率分别为35％、49％，联合治疗的抑瘤率可达62％，终点肿瘤重量见表2。
以上结果提示PD-L1纳米抗体和TLR7激动剂联合治疗具有显著的协同抗肿瘤作用。
表2：终点肿瘤重量
Figure PCTCN2022080953-appb-000009
实施例6：PD-L1和TLR7双靶向纳米抗体偶联药物的制备
将SZU-101、EDCI和NHS溶于DMSO中，室温下搅拌3h，LC-MS监测反应进程，反应完全后，加入十倍量的双蒸水，抽滤，真空干燥即可得到SZU-101-NHS活性酯。
Figure PCTCN2022080953-appb-000010
之后将活化酯用DMSO溶解，抗体和小分子按照1：10的摩尔比剂量反应，投入一定量的小分子活化酯至Nb16中，4℃搅拌反应4小时。反应结束后，在混合物中加入PBS混合，用10kD的生物滤膜过滤除去小分子，得新型偶联化合物Nb16-SZU-101。反应制得的偶联化合物，先将抗体变性打开二硫键，之后用XevoG2XSQTOF质谱仪鉴定样品，主要根据新型偶联化合物比未偶联抗体增加的分子量计算偶联度。
如图4所示，质谱鉴定纳米获得的纳米抗体偶联物的偶联度为4.5。
通过上述方法，制备了PD-L1和TLR7双靶向纳米抗体偶联药物，命名为Nb16-SZU-101(图4A)。
实施例7：PD-L1和TLR7双靶向纳米抗体偶联药物的体外活性测定
7.1 PD-L1和TLR7双靶向纳米抗体偶联药物的PD-L1结合活性测定
消化处理稳转细胞株HEK293T/mPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×10 6细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×10 5细胞/样品；样品中分别加入不同的浓度的偶联化合物Nb16-SZU-101和裸抗Nb16，浓度分别为100、50、25、12.5、6.25、3.12、1.56、0.78、0.39、0.20、0.10、0.05、0.02、0.01、0.006、0.003、0.0008μg/mL，共17个浓度梯度；4℃冰箱孵育20min；离心，去上清， 用PBS洗一遍；用稀释后抗体anti-mouse IgG Fc(PE)作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到Nb16-SZU-101的EC50值曲线，如图5所示。
实验结果提示，SZU-101的偶联并未改变原PD-L1纳米抗体的结合活性。
7.2 PD-L1和TLR7双靶向纳米抗体偶联药物的PD-1/PD-L1阻断活性测定
消化处理稳转细胞株HEK293T/mPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×10 6细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×10 5细胞/样品；样品中分别加入mouse PD-1-biotin蛋白(浓度为20μg/mL)和不同的浓度的偶联化合物Nb16-SZU-101和裸抗Nb16，浓度分别为100、50、25、12.5、6.25、3.12、1.56、0.78、0.39、0.20、0.10、0.05、0.02、0.01、0.006、0.003、0.0008μg/mL，共17个浓度梯度；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体SA-PE作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到Nb16-SZU-101的IC50曲线，如图6所示。
实验结果提示，SZU-101的偶联并未改变原PD-L1纳米抗体的阻断活性。
实施例8：PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性评价
8.1双靶向纳米抗体偶联化合物和联合给药组的体内抗肿瘤作用
在IFN-γ诱导高表达PD-L1的CT26皮下荷瘤BALB/c小鼠模型中，将小鼠随机分为4组，分别为mFc组、Nb16组、Nb16/SZU-101联合给药组以及Nb16-SZU-101纳米抗体偶联药物组，其中mFc或Nb16或Nb16-SZU-101的给药剂量为10mg/kg、给药方式为腹腔给药，SZU-101的给药剂量为0.5mg/kg、给药方式为腹腔给药；分别于Day 2、Day 6、Day 10、Day 13给药，共给药4次。每周2-3次测量肿瘤大小，绘制肿瘤生长曲线；并在Day 14对小鼠进行解剖称重以及拍照。肿瘤生长曲线、终点瘤重和终点肿瘤照片如图7所示。
实验结果表明，本发明中纳米抗体偶联药物Nb16-SZU-101处理组具有显著提高的抗肿瘤效应，该模型中，单独给药组Nb16的抑瘤率为30％，联合给药组Nb16/SZU-101的抑瘤率为39％，纳米抗体偶联药物组的抑瘤率可达81％，终点肿瘤重量见表3。无论相对于单独给药组还是相对于联合给药组，纳米抗体偶联药物组均可显著抑制肿瘤生长。
需要说明的是，同单独给药组Nb16相比，联合给药组并没有显著抑制肿瘤生长，可能是由于联合给药组中SZU-101的剂量降低，且为了和偶联组相一致，给药方式换为腹腔给药，而腹腔给药相对瘤周给药利用度更低等原因导致。
表3：终点肿瘤重量
Figure PCTCN2022080953-appb-000011
Figure PCTCN2022080953-appb-000012
8.2未诱导(低表达)PD-L1及诱导(高表达)PD-L1的CT26皮下荷瘤BALB/c小鼠模型
在图8A中，已表明未诱导的CT26细胞表面PD-L1表达量很低约为17.3％，只有用IFN-γ诱导后PD-L1表达量才会提高至98.1％，因此，我们研究并比较了偶联化合物分别在未诱导以及诱导的CT26小鼠肿瘤模型中的作用。
两个模型中分别将诱导前后的CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中构建荷瘤小鼠模型，接种量为1×10 6细胞/只；两个模型均将小鼠随机分为2组，每组6只，即：空白对照组mFc(浓度为10mg/kg)、以及偶联化合物Nb16-SZU-101组(浓度为10mg/kg)；给药剂量为200μL，给药方式均为腹腔给药；分别在Day 2、Day 5、Day 8、Day 11给药，共给药4次；每周2-3次测量肿瘤大小，绘制肿瘤生长曲线；并在Day 12对小鼠进行解剖称重以及拍照。
从肿瘤生长曲线的结果显示：同阴性对照相比，在CT26诱导前后的两个肿瘤模型中，偶联化合物均可显著抑制肿瘤生长(图8B)，其中未诱导的CT26模型中抑制率为78.9％，而诱导后的CT26模型中抑制率为83.6％，两个模型中偶联化合物的肿瘤抑制效果并没有明显差异。此外，终点肿瘤重量(图8C)和终点肿瘤照片(图8D)的结果，也均同终点肿瘤体积结果基本一致。证实了偶联化合物Nb16-SZU-101对PD-L1低表达和高表达的肿瘤均发挥显著抑制效果。
这提示本发明提供的PD-L1和TLR7双靶向纳米抗体偶联药物不仅对高表达PD-L1的“热”肿瘤有显著抗肿瘤作用，也可以用于PD-L1低表达的“冷”肿瘤的抗肿瘤治疗。
8.3未诱导低表达PD-L1的B16-F10皮下荷瘤C57BL/6模型
基于以上PD-L1和TLR7双靶向纳米抗体偶联药物对低表达PD-L1的CT26肿瘤的抑制作用，本发明进一步评价了PD-L1和TLR7双靶向纳米抗体偶联药物在低表达PD-L1的C57BL/6小鼠B16F10皮下荷瘤模型中的作用。
将6-8周龄的C57BL/6小鼠随机分为2组，分别为mFc组和纳米抗体偶联药物Nb16-SZU-101组。在小鼠腋下皮下种植1×10 6个未诱导低表达PD-L1的B16-F10细胞(图9A)。分别用mFc、纳米抗体偶联药物Nb16-SZU-101给药；对照药和Nb16-SZU-101的给药剂量为10mg/kg、体积200μL；各组给药方式为腹腔注射。在种植肿瘤后的第2、5、8、11天各组分别给药，共给药4次。Day12将小鼠安乐死。肿瘤生长曲线、终点瘤重和终点肿瘤照片如图9B-D所示。
实验结果表明，意外地，在该低表达PD-L1的肿瘤模型中，本发明中纳米抗体偶 联药物Nb16-SZU-101处理组具有显著提高的抗肿瘤效应，该模型中，肿瘤抑制率约68.2％，这提示本发明提供的PD-L1和TLR7双靶向纳米抗体偶联药物可以用于PD-L1低表达的肿瘤的抗肿瘤治疗。
采用的靶向PD-L1的治疗药物或方案通常仅能针对PD-L1高表达的肿瘤，而无法有效用于PD-L1低表达或中表达的肿瘤。因此，本发明的双靶点纳米抗体偶联药物居然可用于治疗PD-L1低表达或中表达的肿瘤，这是很出乎意料的。
8.4早期/晚期CT26皮下荷瘤BALB/c小鼠模型
基于双靶点纳米抗体偶联药物优越的抗肿瘤药效，我们进一步评价了其在CT26早期模型(肿瘤体积<50mm 3)以及晚期模型(肿瘤体积>200mm 3)中的肿瘤抑制作用。
两个模型中分别将未诱导的CT26肿瘤细胞皮下接种至雌性BALB/c小鼠中构建荷瘤小鼠模型，接种量为1×10 6细胞/只；两个模型均将小鼠分为2组，每组8只，即：空白对照组mFc(浓度为10mg/kg)、以及偶联化合物Nb16-SZU-101组(浓度为10mg/kg)；给药剂量为200μL，给药方式均为腹腔给药。早期模型中(肿瘤体积<50mm 3)，在Day4、Day 7、Day 10、Day 13、Day 16给药，共给药5次；晚期模型中(肿瘤体积>200mm3)，在Day9、Day 12、Day 15、Day 17给药，共给药4次。每周2-3次测量肿瘤大小，绘制肿瘤生长曲线。
从肿瘤生长曲线的结果显示：同阴性对照相比，在早期/晚期CT26两种肿瘤模型中，偶联化合物均可显著抑制肿瘤生长，其中早期模型中抑制率为80.3％(图10A)，晚期模型中抑制率为78.9％(图10C)；此外，在两个模型中，双靶点纳米抗体偶联化合物均可显著延长生存期(图10B,D)。而且，在早期模型中，偶联化合物治疗会使高达62.5％的荷瘤小鼠出现肿瘤消退(图10B)；在晚期模型中，偶联化合物用药会使12.5％的荷瘤小鼠出现肿瘤消退(图10D)。小鼠肿瘤消退1周后，对其进行CT26肿瘤细胞的再荷瘤，小鼠在40天内均不再生长肿瘤(图10E)，表明偶联化合物诱导了有效的抗肿瘤免疫记忆，以上结果是令人振奋的。
实施例9：PD-L1和TLR7双靶向纳米抗体偶联药物给药的肿瘤免疫分型分析
在实施例8.1的小鼠解剖终点时，取皮下肿瘤，取150mg肿瘤组织剪碎成肉泥状，于透明质酸酶和胶原酶Ⅳ中37℃下180rpm震荡消化1.5h，处理成单细胞悬液。预处理后的细胞悬液分为两份，一份用于巨噬细胞和树突状细胞的免疫分型分析，一份用于T细胞和NK细胞的免疫分型分析。
巨噬细胞和树突状细胞样品悬液处理：细胞用2-4mL无菌红细胞裂解液处理8min并用PBS缓冲液终止，去除样品悬液中的红细胞及碎片；细胞样品清洗待用。
T细胞和NK细胞样品悬液处理：细胞用淋巴细胞分离液离心处理，获取淋巴细胞分离层，即为肿瘤淋巴细胞悬液，清洗待用。
将巨噬细胞和树突状细胞样品和T细胞和NK细胞样品用Fc受体封闭液4℃封闭1小时，去除Fc受体带来的非特异性染色；进行细胞表面蛋白染色，具体的：M1巨噬细胞(FITC Rat Anti-Mouse CD45、PE-Cyanine7 Rat Anti-Mouse F4/80和APC Rat anti-Mouse CD86)，M2巨噬细胞(FITC Rat Anti-Mouse CD45和PE-Cyanine7 Rat Anti-Mouse F4/80)，树突状细胞(PE-Cy7 Anti-Mouse CD11c、BB515 Rat Anti-Mouse I-A/I-E、PE Hamster Anti-Mouse CD80和APC Rat anti-Mouse CD86)，CD4+T细胞和CD8+T细胞(FITC Hamster Anti-Mouse CD3e、PerCP-Cy TM5.5 Rat Anti-Mouse CD4和APC Rat Anti-Mouse CD8a)，Treg细胞(FITC Hamster Anti-Mouse CD3e、PerCP-Cy TM5.5 Rat Anti-Mouse CD4和PE Rat Anti-Mouse CD25)，NK细胞(CD3-FITC、CD49b-APC、CD69-PE)，随后于冰上染色20min；接着进行胞内蛋白染色，在细胞经受固定破膜处理后，对细胞进行破膜染色，具体的：M2巨噬细胞(Ms CD206 Alexa 647和ANTI-MOUSE LAP PE)染色，含有颗粒酶的功能性T细胞(ANTI-MOUSE GRANZYME B(NGZB)PE)，含有IFN-γ的功能性T细胞(PE Rat Anti-Mouse IFN-γ)，Treg细胞(ANTI-MOUSE/RAT FOXP3 APC)，随后冰上染色20min；清洗后重悬样品细胞液，流式细胞仪上机检测。
流式免疫分型结果如图11所示。
结果表明，与同型对照mFc组相比，PD-L1和TLR7双靶向纳米抗体偶联药物Nb16-SZU-101给药促进了树突状细胞的成熟。同时，PD-L1和TLR7双靶向纳米抗体偶联药物显著增加了肿瘤浸润细胞毒性细胞(CD8+T细胞和NK细胞)的功能，促进了它们的颗粒酶B和IFN-γ的表达。另外，PD-L1和TLR7双靶向纳米抗体偶联给药可促使肿瘤相关巨噬细胞重极化，减少TGF-β+巨噬细胞的浸润。在Nb16-SZU-101治疗组中，IFN-γ+CD4+T细胞的浸润也显著增加。
意外的是，PD-L1和TLR7双靶向纳米抗体偶联药物给药促进了瘤内微环境中巨噬细胞表达PD-L1(图11)，这可以使得PD-L1和TLR7双靶向纳米抗体偶联药物响应低表达PD-L1分子的“冷”肿瘤，达到更广泛的响应，且具有更佳的抗肿瘤药效。此外，在肿瘤微环境中，这些受本发明偶联物激活的免疫细胞(包括CD4+T细胞以及CD8+T细胞等)，其分泌IFN-γ的免疫细胞亚型增多，而IFN-γ是诱导PD-L1表达最常见的细胞因子之一，因此又可进一步促进肿瘤细胞表达PD-L1，从而使得“冷”肿瘤转变为“热”肿瘤。
综上所述，PD-L1和TLR7双靶向纳米抗体偶联药物靶向肿瘤免疫微环境，可重塑肿瘤免疫微环境，协调先天免疫和适应性免疫来发挥抗肿瘤活性。
实施例10：免疫细胞删除分析确定PD-L1和TLR7双靶向纳米抗体偶联药物药效发挥的免疫基础
将6-8周龄的BALB/c小鼠随机分为6组，每组6只动物。对应分组分别为：mFC组、 Nb16-SZU-101组、Nb16-SZU-101+anti-CD4组、Nb16-SZU-101+anti-CD8组、Nb16-SZU-101+anti-NK1.1组、Nb16-SZU-101+氯磷酸脂质体组。mFc和Nb16-SZU-101的给药程序和实施例8.1一致。
免疫细胞删除方法为：分别于Day 3(200ug/只)/Day 4(100ug/只)/Day 5(100ug/只)腹腔注射抗体药(anti-CD4、anti-CD8、anti-NK1.1)删除对应的免疫细胞(CD4+T细胞、CD8+T细胞、NK细胞)，或者腹腔注射氯磷酸-脂质体去除巨噬细胞。
Day14将小鼠安乐死。肿瘤生长曲线和终点肿瘤照片如图12所示。
结果表明，有趣的是，CD8+T细胞和NK细胞的删除极大地影响了PD-L1和TLR7双靶向纳米抗体偶联药物的药效发挥，而CD4+T细胞和巨噬细胞的删除对PD-L1和TLR7双靶向纳米抗体偶联药物的药效发挥影响不大，这说明本发明提供的PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性主要是通过CD8+T细胞和NK细胞发挥作用，杀伤性细胞在药效发挥中无可替代。
实施例11：抗人PD-L1的纳米抗体筛选、表达
如实施例1所述，将human PD-L1蛋白表达获取并免疫骆驼后，构建文库并进行文库淘选，ELISA鉴定获取11株候选阳性克隆。
如实施例1所述，将候选纳米抗体序列同源重组至pFUSE-mIgG2b-Fc和pFUSE-hIgG1-Fc载体中，后利用哺乳动物细胞HEK293F表达候选纳米抗体。
本发明中获得11株优选的抗人PD-L1纳米抗体，分别为h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、h_Nb12、h_Nb13、h_Nb19、h_Nb26、h_Nb30。
其中，h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、h_Nb12、h_Nb13、h_Nb19、h_Nb26、h_Nb30的VHH序列分别如SEQ ID NO.:21、25、29、33、37、40、44、48、52、56、59所示，CDR部分见表4。
表4.抗人PD-L1抗体的VHH及CDR序列
Figure PCTCN2022080953-appb-000013
Figure PCTCN2022080953-appb-000014
Figure PCTCN2022080953-appb-000015
实施例12：抗人PD-L1的纳米抗体的初步体外活性评价
12.1候选抗人PD-L1的纳米抗体的human PD-L1结合活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×10 6细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×10 5细胞/样品；样品中分别加入不同的抗人PD-L1的候选纳米抗体(浓度为10μg/ml)；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-human IgG Fc(FITC)作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到候选纳米抗体与human PD-L1的结合活性，如图13所示。
实验结果提示，候选纳米抗体h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、 h_Nb12、h_Nb13、h_Nb26共9株纳米抗体均与human PD-L1保持良好的结合活性。
12.2候选抗人PD-L1的纳米抗体的human PD-1/PD-L1阻断活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×10 6细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×10 5细胞/样品；样品中分别加入human PD-1-his蛋白(浓度为1μg/mL)和抗人PD-L1的候选纳米抗体(浓度为10μg/ml)；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-his-APC作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到候选纳米抗体的PD-1/PD-L1阻断活，如图14所示。
实验结果提示，候选纳米抗体h_Nb1、h_Nb2可有效阻断PD-1/PD-L1的结合活性,具有明显的阻断效果。
实施例13：抗人PD-L1的纳米抗体的体外活性评价
13.1候选抗人PD-L1的纳米抗体与human PD-L1结合的EC 50活性测定
如实施例12.1所述，消化处理稳转细胞株HEK293T/hPD-L1制备细胞样品；样品中分别加入不同浓度的纳米抗体h_Nb1或h_Nb2或阳性对照抗体KN035；孵育离心结束后，以anti-human IgG Fc(FITC)作为二抗，孵育染色；后用流式细胞仪上机检测，得到候选纳米抗体与human PD-L1结合的EC 50值。
13.2候选抗人PD-L1的纳米抗体阻断human PD-1/PD-L1结合的IC 50活性测定
如实施例12.2所述，消化处理稳转细胞株HEK293T/hPD-L1制备细胞样品；样品中分别加入不同浓度的纳米抗体h_Nb1或h_Nb2或阳性对照抗体KN035，以及human PD-1-his蛋白(浓度为1μg/mL)；孵育离心结束后，以anti-his-APC作为二抗，孵育染色；后用流式细胞仪上机检测，得到候选纳米抗体阻断human PD-1/PD-L1结合的IC 50值。
实施例14：抗人PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性评价
如实施例6所述方法，制备获取抗人PD-L1和TLR7双靶向纳米抗体偶联药物h_Nb1-SZU-101和h_Nb2-SZU-101。
为进一步评价抗人PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性，我们采用PD-1/PD-L1双人化BALB/c小鼠建立的CT26/hPD-L1肿瘤模型，其中给药剂量为10mg/kg、给药方式为腹腔给药。每周2-3次测量肿瘤大小，绘制肿瘤生长曲线。
实施例15：抗人PD-L1的纳米抗体的人源化
利用CDR Grafting方法，将人抗体FR替换驼抗体FR，以达到降低免疫原性的目的。首先对候选抗体进行同源模建，鉴别得到关键氨基酸残基位点，而后以候选纳米抗体序列为模板在结构数据库中进行同源结构的搜索，选择最优结构序列进行序列替 换最终得到人源化抗体序列,同时需考虑保留框架区潜在影响CDR作用的关键位点。
本发明中分别对获得的11株优选的抗人PD-L1纳米抗体进行人源化，其中，人源化抗体后的对应序列如下所示：
h_Nb1人源化后序列分别为h_Nb1_1、h_Nb1_2、h_Nb1_3、h_Nb1_4、h_Nb1_5；
h_Nb2人源化后序列分别为h_Nb2_1、h_Nb2_2、h_Nb2_3、h_Nb2_4、h_Nb2_5；
h_Nb4人源化后序列分别为h_Nb4_1、h_Nb4_2；
h_Nb5人源化后序列分别为h_Nb5_1、h_Nb5_2、h_Nb5_3；
h_Nb6人源化后序列分别为h_Nb6_1、h_Nb6_2；
h_Nb9人源化后序列分别为h_Nb9_1、h_Nb9_2；
h_Nb12人源化后序列分别为h_Nb12_1、h_Nb12_2；
h_Nb13人源化后序列分别为h_Nb13_1、h_Nb13_2；
h_Nb19人源化后序列分别为h_Nb19_1、h_Nb19_2；
h_Nb26人源化后序列分别为h_Nb26_1、h_Nb26_2；
h_Nb30人源化后序列分别为h_Nb30_1、h_Nb30_2。
人源化后抗体序列见表5。
表5.抗人PD-L1抗体人源化后的VHH序列
Figure PCTCN2022080953-appb-000016
Figure PCTCN2022080953-appb-000017
Figure PCTCN2022080953-appb-000018
实施例16：纳米抗体CDR亲和力成熟优化
将根据以上实施例优选的特异性结合人PD-L1纳米抗体的全长氨基酸序列(SEQ ID No.21和SEQ ID No.25)，CCG法注释抗体CDR区和FR区；通过抗体同源模建，选择合适的FR区和CDR区模板，构建并选择最优的纳米抗体三维蛋白结构；从PDB蛋白数据库中获取人PD-L1蛋白晶体结构，通过蛋白复合物同源模建和蛋白-蛋白分子对接方法，获取优选的PD-L1纳米抗体-PD-L1蛋白复合物结构候选库；依据优选抗体的PD-1/PD-L1体外竞争结合特征，优选合适的对接角度，确定最优的对接构象；对选取的纳米抗体-PD-L1蛋白复合物结构分析相互作用关键位点，关注范德华力、氢键、离子键、疏水作用等；重点关注纳米抗体与PD-L1上作用位点的亲和力能量值较低的作用位点对，作为亲和力成熟点突变对象；选取符合以上条件的纳米抗体-PD-L1相互作用残基对，将纳米抗体的对应氨基酸位点进行点突变，分别测试突变为其他19种天然氨基酸，分析突变后位点与PD-L1上对应位点的相互作用力改变，选取使得亲和力显著提高的突变方式；将纳米抗体CDR区上与PD-L1蛋白存在弱相互作用的位点均进行以上点突变筛选，选取优良的突变组合；计算机水平筛选的候选亲和力成熟方案产生的序列，经由基因合成、克隆、转染和蛋白表达纯化获取候选抗体，通过ELISA方法，比较候选抗体和原抗体(SEQ ID No.21和SEQ ID No.25)与PD-L1的亲和力水平和阻断PD-1/PD-L1活性；最终确定优选抗体的CDR区亲和力成熟方案，亲和力成熟后抗体的序列为SEQ ID No.92–SEQ No.117。其中，SEQ ID NO.92-106所示的序列对应的原抗体为h_Nb1VHH(SEQ ID NO.21)；SEQ ID NO.107-117所示的序列对应的原抗体为h_Nb2VHH(SEQ ID NO.25)。
表6.抗人PD-L1抗体亲和力成熟后的VHH序列
Figure PCTCN2022080953-appb-000019
Figure PCTCN2022080953-appb-000020
Figure PCTCN2022080953-appb-000021
Figure PCTCN2022080953-appb-000022
讨论
现有的PD-L1抗体对高免疫原性的“热”肿瘤和PD-L1表达水平高的肿瘤的靶向和抑制作用较好，对于低免疫原性和低表达PD-L1的肿瘤抑制效果较差。因此，联合治疗方案可以立足于提高肿瘤的免疫原性和PD-L1的表达水平，以提高治疗的效果和响应率。
科学合理且安全有效的联合治疗方案同样可以指导新型药物分子的设计，基于PD-L1纳米抗体和TLR7激动剂联合治疗可以显著抑制肿瘤生长，开发了PD-L1和TLR7双靶向纳米抗体偶联药物，达到了优于联合治疗的药效，可以协调统筹先天免疫和适应性免疫应答，靶向并重塑肿瘤免疫微环境，提高肿瘤组织的PD-L1表达水平，使得在“冷”肿瘤和低表达PD-L1的肿瘤模型中同样表现出极其显著的抗肿瘤活性和响应率，显示了其临床应用价值。
本发明首次提供了一种PD-L1和TLR7双靶向纳米抗体偶联药物，可后续进行肿瘤免疫治疗药物开发，尤其适用于低免疫原性和/或低表达PD-L1的肿瘤。
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 (14)

  1. 一种抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的抗体-药物偶联物结构如式Ⅰ所示：
    Ab-(J-U)n    (Ⅰ)
    式中，
    Ab为PD-L1抗体；
    U各自独立地为TLR激动剂；
    J为化学键或连接子；
    n为0或正整数；
    “-”为化学键或接头或连接子。
  2. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的PD-L1抗体为PD-L1纳米抗体或其衍生抗体，其中，所述的PD-L1纳米抗体特异性结合PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组：
    (1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
    (2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
    (3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
    (4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
    (5)SEQ ID NO：18所示的CDR1、SEQ ID NO：19所示的CDR2，SEQ ID NO：20所示的CDR3；
    (6)SEQ ID NO：22所示的CDR1、SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3；
    (7)SEQ ID NO：26所示的CDR1、SEQ ID NO：27所示的CDR2，SEQ ID NO：28所示的CDR3；
    (8)SEQ ID NO：30所示的CDR1、SEQ ID NO：31所示的CDR2，SEQ ID NO：32所示的CDR3；
    (9)SEQ ID NO：34所示的CDR1、SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3；
    (10)SEQ ID NO：22所示的CDR1、SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3；
    (11)SEQ ID NO：41所示的CDR1、SEQ ID NO：42所示的CDR2，SEQ ID NO：43所 示的CDR3；
    (12)SEQ ID NO：45所示的CDR1、SEQ ID NO：46所示的CDR2，SEQ ID NO：47所示的CDR3；
    (13)SEQ ID NO：49所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3；
    (14)SEQ ID NO：53所示的CDR1、SEQ ID NO：54所示的CDR2，SEQ ID NO：55所示的CDR3；
    (15)SEQ ID NO：57所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：58所示的CDR3；和
    (16)SEQ ID NO：60所示的CDR1、SEQ ID NO：61所示的CDR2，SEQ ID NO：62所示的CDR3。
  3. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的TLR激动剂为TLR7激动剂。
  4. 如权利要求3所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的TLR7激动剂包括：SZU-101：
    Figure PCTCN2022080953-appb-100001
  5. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的抗体-药物偶联物或其药学上可接受的盐用于制备一种组合物或制剂，所述组合物或制剂用于：
    (a)促进树突状细胞的成熟；
    (b)增加肿瘤浸润细胞毒性细胞(CD8+T细胞和NK细胞)的功能；
    (c)促进肿瘤浸润细胞毒性细胞颗粒酶B和IFN-γ的表达；
    (d)促使肿瘤相关巨噬细胞重极化；
    (e)减少TGF-β+巨噬细胞的浸润；
    (f)促进IFN-γ+CD4+T细胞的浸润；
    (g)促进瘤内巨噬细胞表达PD-L1；
    (h)靶向并重塑肿瘤免疫微环境；
    (i)提高肿瘤细胞的PD-L1水平；和/或
    (j)用于治疗中表达或低表达PD-L1的肿瘤。
  6. 一种药物组合物，其特征在于，所述药物组合物包含：
    (a)如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐；
    (b)药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述的药物组合物用于治疗PD-L1低表达的肿瘤。
  8. 一种预防或***的方法，其特征在于，向有需要的受试者施用如权利要求1所述的纳米抗体偶联药物。
  9. 一种PD-L1纳米抗体，其特征在于，所述PD-L1纳米抗体特异性结合PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
    (1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
    (2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
    (3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
    (4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
    (5)SEQ ID NO：18所示的CDR1、SEQ ID NO：19所示的CDR2，SEQ ID NO：20所示的CDR3；
    (6)SEQ ID NO：22所示的CDR1、SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3；
    (7)SEQ ID NO：26所示的CDR1、SEQ ID NO：27所示的CDR2，SEQ ID NO：28所示的CDR3；
    (8)SEQ ID NO：30所示的CDR1、SEQ ID NO：31所示的CDR2，SEQ ID NO：32所示的CDR3；
    (9)SEQ ID NO：34所示的CDR1、SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3；
    (10)SEQ ID NO：22所示的CDR1、SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3；
    (11)SEQ ID NO：41所示的CDR1、SEQ ID NO：42所示的CDR2，SEQ ID NO：43所示的CDR3；
    (12)SEQ ID NO：45所示的CDR1、SEQ ID NO：46所示的CDR2，SEQ ID NO：47所示的CDR3；
    (13)SEQ ID NO：49所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3；
    (14)SEQ ID NO：53所示的CDR1、SEQ ID NO：54所示的CDR2，SEQ ID NO：55所示的CDR3；
    (15)SEQ ID NO：57所示的CDR1、SEQ ID NO：50所示的CDR2，SEQ ID NO：58所示的CDR3；和
    (16)SEQ ID NO：60所示的CDR1、SEQ ID NO：61所示的CDR2，SEQ ID NO：62所示的CDR3。
  10. 一种药盒，其特征在于，所述的药盒包括：
    (1)第一容器，以及位于所述第一容器内的如权利要求9所述的PD-L1纳米抗体，以及药学上可用的载体；
    (2)第二容器，以及位于所述第二容器内的TLR7激动剂，以及药学上可用的载体；
    以及(3)任选的使用说明书。
  11. 一种免疫偶联物，其特征在于，所述的免疫偶联物含有：
    (a)如权利要求9所述的PD-L1纳米抗体；和
    (b)其他偶联部分。
  12. 一种融合蛋白，其特征在于，所述的融合蛋白包含：
    (a)如权利要求9所述的PD-L1纳米抗体；和
    (b)任选的具有治疗功能的多肽分子和蛋白片段。
  13. 一种多特异性抗体，其特征在于，所述的多特异性抗体包含：
    (a)如权利要求9所述的PD-L1纳米抗体；和
    (b)任选的靶向第二抗原的抗体分子。
  14. 一种制备权利要求1所述的抗体-药物偶联物的方法，其特征在于，所述方法包括步骤：
    配置反应体系，所述反应体系中包括抗体和游离的药物分子，然后进行偶联反应，从而制得所述抗体-药物偶联物，其中，所述药物分子包括TLR激动剂、接头。
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