WO2022057875A1 - 靶向4-1bb的单域抗体、其融合蛋白、药物组合物及用途 - Google Patents

靶向4-1bb的单域抗体、其融合蛋白、药物组合物及用途 Download PDF

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WO2022057875A1
WO2022057875A1 PCT/CN2021/118955 CN2021118955W WO2022057875A1 WO 2022057875 A1 WO2022057875 A1 WO 2022057875A1 CN 2021118955 W CN2021118955 W CN 2021118955W WO 2022057875 A1 WO2022057875 A1 WO 2022057875A1
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fusion protein
cancer
domain antibody
amino acid
antibody
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PCT/CN2021/118955
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English (en)
French (fr)
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翟天航
缪小牛
徐祎凤
王超
曾竣玮
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普米斯生物技术(珠海)有限公司
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Priority to US18/245,691 priority Critical patent/US20230357422A1/en
Priority to KR1020237012083A priority patent/KR20230070238A/ko
Priority to CN202180062285.XA priority patent/CN116490206A/zh
Priority to EP21868697.0A priority patent/EP4215547A1/en
Priority to JP2023517935A priority patent/JP2023542900A/ja
Priority to CA3195676A priority patent/CA3195676A1/en
Priority to AU2021342525A priority patent/AU2021342525A1/en
Publication of WO2022057875A1 publication Critical patent/WO2022057875A1/zh

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    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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Definitions

  • the invention belongs to the field of biomedicine, and relates to a single domain antibody targeting 4-1BB, a fusion protein thereof, a pharmaceutical composition and use thereof.
  • 4-1BB The tumor necrosis factor receptor superfamily (4-1BB), also known as CD137, or TNFRF9, is a member of the TNF receptor family.
  • 4-1BB is a type I transmembrane protein with a reading frame of 255 amino acids (NCBI: NP_001552), consisting of an N-terminal signal peptide containing 17 amino acids, an extracellular region of 169 amino acids, a transmembrane region of 27 amino acids, and It consists of a C-terminal intracellular domain of 42 amino acids.
  • 4-1BB molecules are mainly expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils and eosinophils, and endothelial cells of tumor blood vessels have also been reported to express 4-1BB. .
  • 4-1BB molecules can provide costimulatory signals for them.
  • the T cell receptor When the T cell receptor is exposed to the antigen, it will increase the expression of 4-1BB.
  • 4-1BB After 4-1BB binds to the ligand, it will cause the activation of the NF ⁇ B signaling pathway, which will lead to the activation and proliferation of T cells, and 4-1BB can also inhibit the Activates apoptosis.
  • Animal models and in vitro experiments confirmed that anti-4-1BB monoclonal antibody has anti-tumor activity. It can selectively induce the proliferation of CD8+ T cells, upregulate the expression of the pro-inflammatory cytokine IFN ⁇ , and enhance the killing effect of antigen-specific effector T cells, thereby promoting tumor clearance.
  • Anti-4-1BB mAb can also cause the expansion of NK cells and increase the cytotoxic activity of CD8+ T cells through it.
  • Anti-4-1BB antibody can also cause the endothelial cells of tumor cells to upregulate the expression of adhesion molecules and promote the infiltration of activated T lymphocytes into tumor tissue.
  • anti-4-1BB activating antibodies also alleviate autoimmune diseases such as autoimmune encephalomyelitis, lupus-like syndrome and collagen-induced arthritis.
  • 4-1BB is an important potential target in the treatment of tumors and some autoimmune diseases.
  • preclinical animal models such as colorectal cancer, lung cancer, breast cancer, and melanoma
  • the agonist molecule of 4-1BB shows significant antitumor activity as a single drug or in combination with antibodies such as anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-HER-2 (Etxeberria I , et al. ESMO Open 2020;4:e000733.).
  • Two 4-1BB antibodies are currently in clinical trials: Urelumab (BMS-663513, developed by Bristol-Myers Squibb) and Utomilumab (PF-05082566, developed by Pfizer).
  • Urelumab is a potent activator with good activating activity, but also has adverse reactions such as liver toxicity and fatigue; while Utomilumab has advantages in safety, but compared with Urelumab, it has receptor agonistic activity. lower.
  • the inventors have developed an anti-human 4-1BB nanobody through in-depth research and creative work. Specifically, the inventors used the human 4-1BB antigen protein to immunize alpaca (Llama) to obtain a high-quality immune nanobody gene library. Among them, the inventors screened the immune nanobody gene library to obtain two anti-4-1BB nanobodies that can simultaneously bind to human and cynomolgus monkey 4-1BB, and need to be activated by external cross-linking. The sequences of the two nanobodies were then optimized, the genetically engineered mutants were expressed and purified, and further screening was carried out in terms of antibody affinity, cross-binding with cynomolgus monkey 4-1BB, and the activity of activating T cells.
  • the human 4-1BB antigen protein to immunize alpaca (Llama) to obtain a high-quality immune nanobody gene library.
  • the inventors screened the immune nanobody gene library to obtain two anti-4-1BB nanobodies that can simultaneously bind to human
  • One aspect of the present invention relates to an anti-4-1BB single domain antibody comprising a heavy chain variable region comprising CDR1-CDR3, wherein the amino acid sequence of CDR1 is selected from SEQ ID NOs:41 -80, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 81-120, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 121-160.
  • One aspect of the present invention relates to an anti-4-1BB single-domain antibody comprising a heavy chain variable region, the heavy chain variable region comprising the amino acid sequences of CDR1-CDR3 as set forth in items 1-40 below, respectively Either one of:
  • the CDRs of anti-4-1BB single domain antibodies are all defined by the IMGT numbering system, please refer to Ehrenmann F, Kaas Q, Lefranc M P. IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009;38(suppl_1):D301-D307.
  • the 4-1BB refers to human 4-1BB unless otherwise specified.
  • the amino acid sequence of 4-1BB is shown in SEQ ID NO: 168.
  • any one, two, three or all four of the four framework regions of the anti-4-1BB single domain antibody are humanized;
  • the second framework region is humanized; optionally, the first, third or fourth framework region is also modified;
  • the homology between the anti-4-1BB single domain antibody and human is greater than or equal to 75%, greater than or equal to 76%, greater than or equal to 77%, greater than or equal to 78%, greater than or equal to 79%, or greater than or equal to 80%;
  • the amino acid sequence of the framework region 1 of the anti-4-1BB single domain antibody is shown in SEQ ID NO: 170
  • the amino acid sequence of the framework region 2 is shown in SEQ ID NO: 171 or SEQ ID NO: 172
  • the framework The amino acid sequence of region 3 is shown in SEQ ID NO:173
  • the amino acid sequence of framework region 4 is shown in SEQ ID NO:174.
  • the anti-4-1BB single domain antibody, its KD with 4-1BB antigen is less than E-07, less than 5E-08, less than 4E-08, less than 3E-08, or less than 2E-08; preferably, the KD is measured by ForteBio .
  • the anti-4-1BB single domain antibody has a different binding site to the 4-1BB antigen than Urelumbab and Utomilumab.
  • the anti-4-1BB single domain antibody specifically binds to human 4-1BB, while cross-binding to cynomolgus 4-1BB.
  • the anti-4-1BB single domain antibody does not block the binding of 4-1BB ligand to 4-1BB in the native state (eg, in mammals, especially in humans). .
  • amino acid sequence of the anti-4-1BB single domain antibody is shown in any one of SEQ ID NOs: 1-40.
  • Another aspect of the present invention relates to a fusion protein comprising the anti-4-1BB single domain antibody of any one of the present invention, and the Fc fragment of human IgG or the constant region of human IgG.
  • the fusion protein is composed of the anti-4-1BB single domain antibody described in any one of the present invention, and the Fc segment of human IgG or the constant region of human IgG.
  • the fusion protein is composed of the anti-4-1BB single domain antibody described in any one of the present invention, a linker, and the Fc segment of human IgG or the constant of human IgG. district composition.
  • the fusion protein wherein, according to the EU numbering system, the Fc segment of human IgG or the heavy chain constant region of human IgG comprises L234A mutation and L235A mutation; alternatively, the The Fc segment of IgG also contains the G237A mutation.
  • the fusion protein is an anti-4-1BB heavy chain antibody.
  • a heavy chain antibody refers to an antibody without a light chain. It is currently a common name for VHH-Fc class antibodies.
  • the fusion protein wherein,
  • the Fc segment of the human IgG is the Fc segment of human IgG1;
  • the heavy chain constant region of the human IgG is the heavy chain constant region of human IgG1.
  • the fusion protein wherein,
  • the Fc segment of the human IgG is the Fc segment of human IgG1 comprising the L234A mutation and the L235A mutation; optionally, the Fc segment of the human IgG1 further comprises the G237A mutation;
  • amino acid sequence of the Fc segment of human IgG1 is shown in SEQ ID NO:167.
  • the fusion protein wherein,
  • the Fc segment of the human IgG or the constant region of the human IgG is directly linked to the C-terminus of the anti-4-1BB single domain antibody or linked by a linking fragment.
  • the fusion protein wherein,
  • the Fc segment of human IgG1 or the constant region of human IgG1 is directly linked to the C-terminus of the anti-4-1BB single domain antibody or linked through a linking fragment.
  • Yet another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention.
  • the present invention also relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • the present invention also relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
  • Yet another aspect of the present invention relates to a method for preparing the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention, comprising culturing the present invention under suitable conditions Host cells of the invention, and steps for recovering said anti-4-1BB single domain antibody or fusion protein from cell culture.
  • Another aspect of the present invention relates to a conjugate comprising an antibody moiety and a coupling moiety, wherein the antibody moiety is the anti-4-1BB single domain antibody of any one of the present invention or any one of the present invention
  • the coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • Still another aspect of the present invention relates to a kit comprising the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention, or the conjugate of the present invention thing;
  • the kit further comprises a second antibody capable of specifically binding to the single domain antibody or fusion protein; optionally, the second antibody further comprises a detectable label, such as radioisotopes, fluorescent substances, Luminescent substances, colored substances or enzymes.
  • a detectable label such as radioisotopes, fluorescent substances, Luminescent substances, colored substances or enzymes.
  • Yet another aspect of the present invention relates to the use of the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention in the preparation of a kit for detecting The presence or level of 4-1BB in the sample.
  • Still another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention or comprising the conjugate of the present invention drug; optionally, it also includes pharmaceutically acceptable excipients.
  • Another aspect of the present invention relates to the anti-4-1BB single domain antibody of any one of the present invention or the fusion protein of any one of the present invention or the conjugate of the present invention in the preparation of preventing and/or treating malignant Use in medicines for tumors or autoimmune diseases;
  • the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, esophageal cancer and head and neck cancer;
  • the autoimmune disease is selected from autoimmune encephalomyelitis, lupus-like syndrome and collagen-induced arthritis.
  • Yet another aspect of the present invention relates to a method of treating and/or preventing malignant tumor autoimmune diseases, comprising administering to a subject in need thereof an effective amount of the anti-4-1BB single domain of any one of the present invention
  • the steps of the antibody or fusion protein of any one of the present invention or the conjugate of the present invention are provided.
  • the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, esophageal cancer and head and neck cancer;
  • the autoimmune disease is selected from autoimmune encephalomyelitis, lupus-like syndrome and collagen-induced arthritis.
  • the method wherein the step of administering to a subject in need thereof an effective amount of the single domain antibody or fusion protein of any one of the present invention is prior to surgical treatment or After, and/or before or after radiation therapy.
  • the single-administration dose of the single-domain antibody or fusion protein of the present invention is 0.1-100 mg per kilogram body weight, preferably 4.8-24 mg or 1-10 mg; or, the single-domain antibody or fusion protein of the present invention is administered at a single dose of 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg per subject;
  • it is administered every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the mode of administration is intravenous drip or intravenous injection.
  • the anti-4-1BB single domain antibody according to any one of the present invention or the fusion protein according to any one of the present invention or the conjugate of the present invention is used for the treatment and/or prevention of malignant tumor or autoimmunity sexually transmitted diseases;
  • the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, esophageal cancer and head and neck cancer;
  • the autoimmune disease is selected from autoimmune encephalomyelitis, lupus-like syndrome and collagen-induced arthritis.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but linkers that are too short are used to allow for both domains on the same chain Pairing between the domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993;90:6444 -6448 and Poljak RJet al., Structure 1994;2:1121-1123).
  • a fusion protein as described herein is a protein product of co-expression of two genes obtained by DNA recombination.
  • Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art (eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15).
  • isolated refers to artificially obtained from the natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated or isolated
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the term "pharmaceutically acceptable excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (See, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed . Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor);
  • a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications. Determining such effective amounts is well within the ability of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently etc.
  • the present invention also relates to any one of the following items 1 to 11:
  • An anti-4-1BB Nanobody characterized in that the VHH chain of the Anti-4-1BB Nanobody has an amino acid sequence as shown in any of SEQ ID NOs:1-40;
  • any amino acid sequence in the above-mentioned amino acid sequence also includes 1-8 (preferably 1-5, more preferably 1-3) amino acid residues that are optionally added, deleted, modified and/or replaced , and a derived sequence capable of retaining the 4-1BB binding affinity of the 4-1BB Nanobody.
  • a fusion protein is characterized in that, described fusion protein has the structure shown in formula I from N-terminal to C-terminal:
  • Z1 is one or more (preferably 1-2, more preferably 1) VHH chains of the anti-4-1BB Nanobody according to item 1;
  • Z2 is the Fc segment of immunoglobulin
  • L is an optional linker sequence.
  • a polynucleotide wherein the polynucleotide encodes the anti-4-1BB nanobody according to item 1 or the fusion protein according to item 2.
  • An expression vector comprising the polynucleotide according to item 3.
  • a host cell characterized in that the host cell contains the expression vector according to item 4, or the polynucleotide according to item 3 is integrated into its genome.
  • a method for producing anti-4-1BB Nanobody or fusion protein is characterized in that, comprises the steps:
  • immunoconjugate characterized in that, the immunoconjugate contains:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • a pharmaceutical composition characterized in that, comprising:
  • a recombinant protein wherein the recombinant protein has:
  • a protein complex characterized in that, the complex is formed by 4-1BB protein and anti-4-1BB antibody or its antigen-binding fragment through hydrogen bonding and/or hydrophobic interaction;
  • the described hydrogen bonding site includes more than 4 (preferably more than 5, more preferably more than 6, most preferably 7) located in the 4-1BB protein and is selected from the following group of sites: Asp 118, Leu 123, Arg 130, Val 132, Cys 134, Gly 135, and Ser 137;
  • the constituting sites of the hydrophobic interaction interface include more than 2 (preferably more than 3, more preferably more than 4) sites located in the 4-1BB protein selected from the following group: the 123rd position Leu, 124th Val, 133rd Val, and 136th Pro;
  • amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168.
  • the present invention also relates to any one of the following first to fifteenth aspects:
  • an anti-4-1BB Nanobody is provided, the VHH chain of the anti-4-1BB Nanobody has the amino acid sequence shown in any of SEQ ID NOs: 1-40;
  • any amino acid sequence in the above-mentioned amino acid sequence also includes 1-8 (preferably 1-5, more preferably 1-3) amino acid residues that are optionally added, deleted, modified and/or replaced , and a derived sequence capable of retaining the 4-1BB binding affinity of the 4-1BB Nanobody.
  • amino acid sequence of the VHH chain of the anti-4-1BB Nanobody is shown in any one of SEQ ID Nos: 3, 35, 37, 38, 39 or 40.
  • a fusion protein is provided, and the fusion protein has the structure shown in formula I from the N-terminus to the C-terminus:
  • Z1 is one or more (preferably 1-2, more preferably 1) VHH chains of the anti-4-1BB Nanobody according to the first aspect of the present invention
  • Z2 is the Fc segment of immunoglobulin
  • L is an optional linker sequence.
  • the fusion protein is a dimer, and the dimer is formed by disulfide bonds between Fc segments in Z2.
  • the immunoglobulin is IgG1, IgG2, IgG3 or IgG4; preferably IgG1, IgG2 or IgG4.
  • the immunoglobulin is IgG1, or a mutant thereof.
  • the L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , or a combination thereof.
  • amino acid sequence of Z2 is shown in SEQ ID NO: 167.
  • amino acid sequence of the Z2 is identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 167.
  • the said substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, and most preferably 1- 3) amino acids are not identical, wherein the non-identity includes amino acid substitution, deletion or addition.
  • the substantially identical amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 86% identical to the corresponding amino acid sequence. 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96% 97%, at least 98%, or at least 99%.
  • the amino acid sequence of Z1 is as shown in any of SEQ ID NO:1-40, and L is none, and the amino acid sequence of Z2 is as shown in SEQ ID NO:167 shown;
  • any amino acid sequence in the above-mentioned amino acid sequence also includes 1-8 (preferably 1-5, more preferably 1-3) amino acid residues that are optionally added, deleted, modified and/or replaced , and derived sequences capable of retaining 4-1BB binding affinity.
  • the amino acid sequence of Z1 is shown in any of SEQ ID NO: 3, 35, 37, 38, 39 or 40, and L is none, and Z2 is The amino acid sequence is shown in SEQ ID NO: 167;
  • any amino acid sequence in the above-mentioned amino acid sequence also includes 1-8 (preferably 1-5, more preferably 1-3) amino acid residues that are optionally added, deleted, modified and/or replaced , and derived sequences capable of retaining 4-1BB binding affinity.
  • the sequence in the amino acid sequence of the fusion protein, from the N-terminus to the C-terminus, is as follows: the sequence shown in any of SEQ ID NO:1-40 and the sequence shown in SEQ ID NO:167 sequence.
  • the sequence from the N-terminus to the C-terminus is: the sequence shown in any one of SEQ ID NO: 3, 35, 37, 38, 39 or 40, and The sequence shown in SEQ ID NO:167.
  • a polynucleotide encoding the anti-4-1BB nanobody according to the first aspect of the present invention or the fusion protein according to the second aspect of the present invention is provided.
  • the polynucleotide includes DNA or RNA.
  • an expression vector containing the polynucleotide according to the third aspect of the present invention there is provided an expression vector containing the polynucleotide according to the third aspect of the present invention.
  • a host cell contains the expression vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated into its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.
  • an immunoconjugate is provided, the immunoconjugate contains:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the coupling moiety is a drug or a toxin.
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pre- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (eg, bivalent) anti-4-1BB nanobody as described in the first aspect of the present invention, or a fusion as described in the second aspect of the present invention protein.
  • the multivalent refers to the anti-4-1BB nanobody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate, or the anti-4-1BB nanobody according to the present invention
  • the fusion protein of the second aspect refers to the anti-4-1BB nanobody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate, or the anti-4-1BB nanobody according to the present invention
  • the fusion protein of the second aspect refers to the anti-4-1BB nanobody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate, or the anti-4-1BB nanobody according to the present invention
  • the fusion protein of the second aspect refers to the anti-4-1BB nanobody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate, or the anti-4-1BB nanobody according to the present invention
  • the fusion protein of the second aspect refers to the anti-4-1BB nanobody according to the first aspect
  • the anti-4-1BB Nanobody as described in the first aspect of the present invention or the fusion protein as described in the second aspect of the present invention for preparing (a) for detecting 4 - Reagents for 1BB molecules; (b) drugs for the treatment of tumors.
  • the detection includes flow detection and cellular immunofluorescence detection.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a drug for treating tumors, and the tumor is selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck cancer, nasopharyngeal cancer, thyroid tumor, tongue cancer, or a combination thereof.
  • the use is non-diagnostic and non-therapeutic.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes: 6His tag, HA tag, Flag tag, Fc tag, or a combination thereof.
  • the recombinant protein specifically binds to 4-1BB protein.
  • the anti-4-1BB Nanobody according to the first aspect of the present invention the fusion protein according to the second aspect of the present invention, or the immunization according to the seventh aspect of the present invention
  • the reagent, detection plate or kit is used for: detecting 4-1BB protein in the sample;
  • the medicament is used for the treatment or prevention of various hematological tumors and solid tumors.
  • the tumors include: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, adrenal tumor, melanoma , head and neck cancer, nasopharyngeal cancer, thyroid tumor, tongue cancer, or a combination thereof.
  • a method for detecting 4-1BB protein in a sample comprising the steps of:
  • the detection includes qualitative detection and quantitative detection.
  • a method for treating a disease comprising administering the anti-4-1BB nanobody as described in the first aspect of the present invention, as described in the second aspect of the present invention to a subject in need
  • the fusion protein or the immunoconjugate according to the seventh aspect of the present invention comprising administering the anti-4-1BB nanobody as described in the first aspect of the present invention, as described in the second aspect of the present invention to a subject in need.
  • the subject includes a mammal.
  • the mammal is a human.
  • a protein complex formed by the interaction between 4-1BB protein and an anti-4-1BB antibody or an antigen-binding fragment thereof.
  • the interaction includes hydrogen bonding and hydrophobicity.
  • the anti-4-1BB antibody is an anti-4-1BB nanobody.
  • the anti-4-1BB antibody is the anti-4-1BB nanobody according to the first aspect of the present invention.
  • the complex is formed by hydrogen bonding and/or hydrophobic interaction between 4-1BB protein and anti-4-1BB antibody or its antigen-binding fragment;
  • the described hydrogen bonding sites include more than 4 (preferably more than 5, more preferably more than 6, most preferably 7) sites located in the 4-1BB protein selected from the following group: 118 Asp, 123 Leu, 130 Arg, 132 Val, 134 Cys, 135 Gly, and 137 Ser;
  • the constituting sites of the hydrophobic interaction interface include more than 2 (preferably more than 3, more preferably more than 4) sites located in the 4-1BB protein selected from the following group: the 123rd position Leu, 124th Val, 133rd Val, and 136th Pro;
  • amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168.
  • the hydrophobic interaction interface also includes more than 4 (preferably 5 or more, more preferably 6 or more, optimally 7 or more or 8 or more) located on the anti-4- In the 1BB antibody or an antigen-binding fragment thereof, a site selected from the group consisting of: Val at position 32, Ala at position 33, Tyr at position 37, Leu at position 47, Ile at position 52, Tyr at position 97, Tyr at position 102 , and the 115th Trp;
  • amino acid numbering of the anti-4-1BB antibody or its antigen-binding fragment is based on the numbering of SEQ ID NO: 169 or 39.
  • amino acid sequence of the 4-1BB protein in the complex is shown in SEQ ID NO: 168.
  • amino acid sequence of the anti-4-1BB nanobody in the complex is shown in SEQ ID NO: 169 or 39.
  • the hydrogen bond is located at the 118th Asp, the 123rd Leu, the 130th Arg, the 132nd Val, the 134th Cys, the 135th Gly in the 4-1BB protein, and / or 137th Ser;
  • amino acid numbering is based on the numbering of SEQ ID NO: 168.
  • the hydrophobic interaction interface is located at the 123rd Leu, 124th Val, 133rd Val and/or 136th Pro of the 4-1BB protein, and the anti-4-1BB nanometer Val at position 32, Ala at position 33, Tyr at position 37, Leu at position 47, Ile at position 52, Tyr at position 97, Tyr at position 102 and/or Trp at position 115 of the VHH chain of the antibody;
  • amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168
  • amino acid numbering of the VHH chain of the anti-4-1BB Nanobody is based on the numbering of SEQ ID NO: 169 or 39.
  • the term "about” can refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • administration and “administration” are used interchangeably and refer to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including Intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, eg, by injection or infusion.
  • Nanobodies of the present invention As used herein, the terms “Nanobodies of the present invention”, “anti-4-1BB Nanobodies of the present invention”, “4-1BB Nanobodies of the present invention” are used interchangeably and all refer to specific recognition and binding to 4-1BB ( Nanobodies including human 4-1BB). Particularly preferred are Nanobodies whose amino acid sequence of the VHH chain is as shown in any of SEQ ID NOs: 1-40, more preferably the amino acid sequence of the VHH chain is as shown in SEQ ID NO: 3, 35, 37, 38, 39 or Nanobodies shown in any of 40.
  • single domain antibody VHH
  • nanobody cloning the variable region of an antibody heavy chain to construct a single domain antibody consisting of only one heavy chain variable region (VHH), which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 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.
  • 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 regions 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.
  • the present invention also includes cell surface markers or antigens that bind to the anti-4-1BB protein antibody or fragment thereof.
  • 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 4-1BB protein, such as a protein having a heavy chain variable region or peptides. 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), four FR amino acids
  • FRs framework regions
  • FRs framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • 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.
  • Nanobodies also have 4 framework regions (framwork), of which the 1st, 3rd, and 4th framework regions (framework region 1, framework region 3 and framework region 4) have high homology with human antibodies and are modified during the humanization process. It is less necessary to transform the second framework area, namely framework area 2 (framework 2).
  • 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 4-1BB protein-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 of 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 mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide 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 under 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 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 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • fusion protein of the present invention refers to a fusion protein that has both the anti-4-1BB nanobody described in the first aspect of the present invention and the Fc segment of immunoglobulin.
  • a fusion protein is provided, and the nanobody fusion protein has the structure shown in formula I from the N-terminus to the C-terminus:
  • Z1 is one or more VHH chains of the anti-4-1BB Nanobody according to the first aspect of the present invention.
  • Z2 is the Fc segment of immunoglobulin
  • L is an optional linker sequence.
  • the immunoglobulin can be IgG1, IgG2, IgG3 or IgG4 and the like. (preferably IgG1, IgG2 or IgG4), but also different mutants of IgG1.
  • the fusion protein is a dimer formed by disulfide bonds between Fc segments in Z2.
  • the L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , or a combination thereof.
  • amino acid sequence of Z2 is shown in SEQ ID NO:167.
  • amino acid sequence of said Z2 is the same or substantially the same as the amino acid sequence set forth in SEQ ID NO: 167.
  • said substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, most preferably 1-3) amino acids Not identical, wherein said not identical includes amino acid substitutions, deletions or additions.
  • the substantially identical amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 87% identical to the corresponding amino acid sequence. 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the sequence from the N-terminus to the C-terminus is as follows: the sequence shown in any of SEQ ID NO: 1-40 and the sequence shown in SEQ ID NO: 167 the sequence shown;
  • any amino acid sequence in the above-mentioned amino acid sequence also includes 1-8 (preferably 1-5, more preferably 1-3) amino acid residues that are optionally added, deleted, modified and/or replaced , and derived sequences capable of retaining 4-1BB binding affinity.
  • the fusion protein of the present invention has the advantages of high dual-target binding affinity and strong specificity, thereby further enhancing the anti-tumor immune function.
  • 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, and a pharmaceutically acceptable carrier.
  • 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, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind 4-1BB protein molecules, and thus can be used to treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned Nanobody (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient.
  • a safe and effective amount such as 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %
  • pharmaceutically acceptable 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 the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the Nanobody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the anti-4-1BB nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.
  • the anti-4-1BB nanobody of the present invention has good specificity and high titer.
  • the present invention also relates to methods of detecting 4-1BB protein.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of 4-1BB protein in the lysed sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) or fusion protein or detection plate of the present invention.
  • the kit further includes a container, an instruction manual, and a buffer. Wait.
  • the present invention also provides a detection kit for detecting the level of 4-1BB, the kit includes an antibody that recognizes the 4-1BB protein, a lysis medium for dissolving the sample, general reagents and buffers required for the detection, such as various Buffers, detection labels, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the nanobody of the present invention has a wide range of biological application value and clinical application value, and its application involves diagnosis and treatment of 4-1BB-related diseases, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy for 4-1BB.
  • the nanobody of the present invention has a small molecular weight.
  • the nanobody of the present invention can highly specifically bind to human 4-1BB protein, while cross-binding to cynomolgus monkey protein.
  • the Nanobody of the present invention needs to be activated by external cross-linking, and has excellent activity and safety.
  • the Nanobody of the present invention does not block the binding of 4-1BB ligand to 4-1BB in natural state.
  • Urelumab As an agonist molecule against 4-1BB, Urelumab leads to clinical liver toxicity due to its strong activation activity.
  • An important advantage of the antibody of the present invention is that the binding site of 4-1BB is different from that of Urelumbab and Utomilumab, and the activation activity is between Urelumab and Utomilumab.
  • Figure 1A The detection result of the first batch of 4-1BB antibodies of the present invention binding to CHO cells stably expressing human 4-1BB.
  • Figure 1B The detection result of the second batch of 4-1BB antibodies of the present invention binding to CHO cells stably expressing human 4-1BB.
  • Figure 2A The detection result of the first batch of 4-1BB antibodies of the present invention binding to cynomolgus monkey 4-1BB protein.
  • Figure 2B The detection result of the second batch of 4-1BB antibodies of the present invention binding to cynomolgus monkey 4-1BB protein.
  • Figure 3A The detection results of the first batch of 4-1BB antibodies of the present invention activating primary T cells to produce IFN- ⁇ .
  • Figure 3B The detection results of the second batch of 4-1BB antibodies of the present invention to activate primary T cells to produce IFN- ⁇ .
  • Figure 4A The detection results of the genetically engineered progeny molecules of the L-Yr-13&14-16 molecules of the present invention binding to CHO cells stably expressing human 4-1BB.
  • Figure 4B The detection result of the genetically engineered progeny molecule of the A-Na-19 molecule of the present invention binding to CHO cells stably expressing human 4-1BB.
  • Figure 5A The detection results of the genetically engineered progeny molecules of the L-Yr-13&14-16 molecules of the present invention binding to CHO cells stably expressing cynomolgus monkey 4-1BB.
  • Figure 5B The detection result of the genetically engineered progeny molecule of the A-Na-19 molecule of the present invention binding to CHO cells stably expressing cynomolgus monkey 4-1BB.
  • Figure 6A The detection results of the genetically engineered progeny molecules of the L-Yr-13&14-16 molecules of the present invention activating primary T cells to produce IFN- ⁇ .
  • Figure 6B The detection result of the gene-engineered progeny molecule of the A-Na-19 molecule of the present invention activating primary T cells to produce IFN- ⁇ .
  • Figure 7A The detection result of the genetically engineered progeny molecules of the L-Yr-13&14-16 molecules of the present invention blocking the binding of 4-1BB ligands to 4-1BB.
  • Figure 7B The detection results of the binding of L-Yr-13&14-16-1 molecules of the present invention to TNFRSF members.
  • Figure 8 Schematic crystal representation of the 4-1BB-VHH complex of the present invention.
  • Figure 9 Crystal structure of the 4-1BB-VHH complex of the present invention.
  • green is 4-1BB protein; blue is VHH chain of anti-4-1BB nanobody.
  • Figure 10 Hydrogen bonding interaction interface in the crystal structure of the 4-1BB-VHH complex of the present invention.
  • green is 4-1BB protein
  • blue is VHH chain of anti-4-1BB nanobody.
  • Figure 11 Hydrophobic interaction interface in the crystal structure of the 4-1BB-VHH complex of the present invention.
  • green is 4-1BB protein
  • blue is VHH chain of anti-4-1BB nanobody.
  • anti-4-1BB Nanobodies involved and the sequence numbers of their CDRs are shown in Table B below.
  • A-Ye-23 16 56 96 136 A-Ye-25 17 57 97 137 A-Ye-27 18 58 98 138 A-Ye-30 19 59 99 139 A-Ye-33 20 60 100 140 A-Ye-20 twenty one 61 101 141 A-Ye-35 twenty two 62 102 142 L-Yr-13&14-01 twenty three 63 103 143 L-Yr-13&14-02 twenty four 64 104 144 L-Yr-13&14-03 25 65 105 145 L-Yr-13&14-05 26 66 106 146 L-Yr-13&14-06 27 67 107 147 L-Yr-13&14-07 28 68 108 148 L-Yr-13&14-08 29 69 109 149 L-Yr-13&14-09 30 70 110 150 L-Yr-13&14-10 31 71 111 151 L-Yr-13&14-11 32 72 112 152 L-Yr-13&14-12 33 73 113 153 L-
  • the CDRs of each Nanobody are defined by the IMGT numbering system.
  • SEQ ID Nos: 41-160 are CDR sequences, some of which may be the same, and different numbers are used for ease of expression.
  • RNA extraction reagent Trizol purchased from Invitrogen
  • Alpaca total cDNA was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).
  • IgG2 and IgG3 sequences were amplified from cDNA:
  • the first round of PCR products were subjected to agarose gel electrophoresis, and the fragment at 750 bp was recovered by cutting the gel for the second round of VHH sequence amplification.
  • the second round PCR amplification primers are as follows:
  • the target fragment was recovered using a PCR purification kit (purchased from QIAGEN).
  • the linearized yeast display vector and the PCR product of the third round were mixed and electrotransformed into Saccharomyces cerevisiae ( 20828), an anti-4-1BB Nanobody library from two alpacas was constructed respectively.
  • the VHH library constructed in Example 1 was inoculated into SD-CAA expansion medium (1L SD-CAA expansion medium contained 6.7g YNB, 5g casamino acid, 13.62g Na 2 HPO 4 ⁇ 12H 2 O, 7.44 g g NaH 2 PO 4 and 2% glucose) overnight at 30°C, 225 rpm. Take an appropriate amount of yeast cells, remove the medium by centrifugation at 3000rpm ⁇ 5min, resuspend the yeast cells with SD-CAA induction medium, and induce overnight. Measure the library concentration after induction, take an appropriate amount of yeast cells, and remove the medium by centrifugation. The yeast cells were resuspended in 50 ml wash buffer (PBS+0.5%BSA+2mM EDTA), and the supernatant was removed by centrifugation. Resuspend yeast cells with 10 ml of wash buffer.
  • Biotin-labeled 4-1BB protein (final concentration 100 mM) was added, incubated at room temperature for 30 min, the yeast cells were collected by centrifugation, and the yeast was washed three times with 50 ml of washing buffer.
  • the yeast cells were resuspended with 5 ml of wash buffer, 200 ⁇ l of SA magnetic beads (purchased from Miltenyi) were added, and the cells were incubated upside down for 10 min.
  • the yeast and magnetic bead mixture was washed 3 times with wash buffer, and the mixture was applied to an LS column (purchased from Miltenyi). Place the LS column on a magnetic stand and wash with wash buffer to remove non-specifically bound yeast cells. Remove the column from the magnetic stand and add wash buffer to elute the yeast.
  • the eluted yeast was centrifuged and transferred to 200ml SD-CAA amplification medium for amplification.
  • the yeast cells enriched by MACS were inoculated into SD-CAA expansion medium and cultured overnight in shake flasks at 30°C and 225rpm.
  • Use SD-CAA induction medium (1L SD-CAA induction medium containing 6.7g YNB, 5g casein amino acid, 13.62g Na 2 HPO 4 ⁇ 12H 2 O, 7.44g NaH 2 PO 4 and 2% galactose, 2 % raffinose and 0.1% glucose) to resuspend yeast cells and induce overnight.
  • Anti-c-Myc murine antibody purchased from Thermo
  • 100 nM biotin-labeled 4-1BB antigen were added at 1:200 dilution, and incubated at room temperature for 10 min.
  • the yeast liquid enriched by MACS and FACS with high binding ability to 4-1BB antigen was cultured in SD-CAA amplification medium at 30°C and 225 rpm overnight, according to the yeast plasmid extraction kit (purchased from Tiangen) operation to extract yeast plasmids.
  • the plasmid was electroporated into Top10 competent cells (purchased from Tiangen), coated with ampicillin-resistant plates, and cultured at 37°C overnight. Pick a single clone for sequencing to obtain the VHH gene sequence.
  • VHH gene sequence obtained in Example 2 was connected to the Fc segment of human IgG1 (LALA mutation), and the linearized pCDNA3.1 vector was cut with homologous recombinase (purchased from Vazyme) and EcoR I/Not I.
  • the process was as follows Product manual. Homologous recombination products were transformed into Top10 competent cells, coated with ampicillin-resistant plates, cultured at 37°C overnight, and single clones were picked for sequencing.
  • the ExpiCHO TM expression system kit (purchased from Thermo) was used to transfer the plasmid into Expi-CHO cells, and the transfection method was in accordance with the commercial instructions. After 5 days of cell culture, the supernatant was collected using protein A magnetic beads (purchased from Nanjing GenScript Bio Technology Co., Ltd.) sorting method to purify the target protein. The magnetic beads were resuspended (1-4 times the volume of magnetic beads) with an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking.
  • PBS+0.1% Tween 20, pH 7.4 an appropriate volume of binding buffer
  • the samples were placed on a magnetic stand (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer.
  • Add the elution buffer (0.1M sodium citrate, pH 3.2) according to the volume of 3-5 times the volume of the magnetic beads, shake at room temperature for 5-10 minutes, put it back on the magnetic rack, collect the elution buffer, and transfer it to the neutralization buffer that has been added (1M Tris, pH 8.54) in a collection tube.
  • the 4-1BB antibody fusion protein samples obtained after purification were used in Examples 4 to 7 below.
  • CHO cells (CHO-h4-1BB cells) overexpressing human 4-1BB were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) encoding human 4-1BB cDNA cloned into MCS (multiple cloning site).
  • the expanded cultured CHO-h4-1BB cells were adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for later use.
  • Cynomolgus monkey 4-1BB-his protein (purchased from ACRO) was dissolved according to the instructions, diluted to 1 ⁇ g/ml with ELISA coating solution (purchased from Shanghai Sangong), 100 ⁇ l/well was coated on ELISA plate, overnight at 4°C, washed with PBST 3 times, 5% BSA (purchased from Shanghai Shenggong) was blocked at room temperature for 1 hour. Discard the coating solution, add 100 ⁇ l/well of 1% BSA serially diluted 4-1BB antibody, 200nM 3-fold dilution for a total of 12 points, and incubate at room temperature for 2 hours.
  • PBST was washed three times, and 100 ⁇ l/well of goat anti-human IgG-Fc-HRP (purchased from abcam) diluted with 1% BSA was added, and incubated at room temperature for 1 hour.
  • PBST was washed 3 times, 100 ⁇ l/well was added with ELISA color developing solution (purchased from Soleibo) for reaction at room temperature for 3 minutes, 50 ⁇ l/well was added with ELISA stop solution (purchased from Soleibo), and the absorbance value at 450nm was read.
  • OKT-3 antibody (purchased from Biolegend) was diluted to 1 ⁇ g/ml with sterile PBS (purchased from Hyclone), 50 ⁇ l/well was coated on a 96-well cell culture flat bottom plate (purchased from Thermo), and 50 ⁇ l/well was added with PBS serially diluted Anti-4-1BB antibody, incubated at 37°C for two hours.
  • Resuscitate cryopreserved human PBMCs purchasedd from Shanghai Saili
  • isolate T cells with a human T cell isolation and purification kit purchasedd from Stemcell
  • X-VIVO15 medium purchasedd from LONZA
  • the coating solution was discarded, washed twice with PBS, discarded PBS, and 200 ⁇ l/well of the above T cell suspension was added.
  • the cells were cultured at 37°C and 5% CO 2 for 5 days, and the supernatant was collected to detect the IFN- ⁇ content.
  • the partially purified samples of the present invention can activate human primary T cells to secrete IFN- ⁇ , and some samples (such as A-Na-16, A-Na-18, A-Na- The activity of 19) was comparable to that of the control antibody Urelumab (US20090068192), and the activities of some samples (eg L-Yr-13&14-16, L-Yr-13&14-17) were between Urelumab and Utomiumab.
  • Example 3 The protein construction, expression and purification methods were the same as those in Example 3.
  • the purified genetically engineered antibody was subjected to human 4-1BB CHO-S cell binding detection, and the detection method was the same as that in Example 4.
  • the results are shown in Fig. 4A and Fig. 4B, the genetically engineered antibody maintained binding activity to human 4-1BB CHO-S cells.
  • Cynomolgus 4-1BB CHO-S cell binding assay was performed on purified genetically engineered antibodies. The results are shown in Figs. 5A and 5B. The results showed that the genetically engineered HZ-L-Yr-13&14-16 antibodies maintained their binding activity to cynomolgus monkey 4-1BB CHO-S cells, while the genetically engineered A-Na-19 antibodies HZ-A-Na The binding activity of -19-1 and HZ-A-Na-19-2 to cynomolgus monkey 4-1BB CHO-S cells decreased significantly.
  • the primary T cell activation assay was performed on the purified genetically engineered antibody, and the detection method was the same as that in Example 7. The results are shown in FIGS. 6A and 6B . The results showed that the genetically engineered HZ-L-Yr-13&14-16-1 and HZ-A-Na-19-1 maintained the activation activity of primary T cells.
  • the expanded cultured CHO-h4-1BB cells were adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for later use.
  • the purified 4-1BB antibody was diluted to 400 nM with PBS, and 50 ⁇ l/well of the above diluted sample was added to the above-mentioned 96-well flow plate with cells, and incubated at 4° C. for 30 min.
  • 50 ⁇ l/well of Biotin-4-1BB ligand protein purchased from ACRO
  • diluted to 1 ⁇ g/ml in PBS was added, and incubated at 4° C. for 30 min.
  • Human 4-1BB-his, GITR-his, OX40-his, CD40-his proteins (purchased from ACRO) were dissolved according to the instructions, diluted to 1 ⁇ g/ml with ELISA coating solution (purchased from Shanghai Sangong), and 100 ⁇ l/well was coated The ELISA plate was placed at 4°C overnight, washed three times with PBST, and 200 ⁇ l/well was added with 5% BSA (purchased from Shanghai Sangong) for blocking at room temperature for 1 hour. Discard the coating solution, add 100 ⁇ l/well of 4-1BB antibody diluted in 1% BSA, 200nM into 3 replicate wells, and incubate at room temperature for 2 hours.
  • PBST was washed three times, and 100 ⁇ l/well of goat anti-human IgG-Fc-HRP (purchased from abcam) diluted with 1% BSA was added, and incubated at room temperature for 1 hour.
  • PBST was washed 3 times, 100 ⁇ l/well was added with ELISA color developing solution (purchased from Soleibo) for reaction at room temperature for 3 minutes, 50 ⁇ l/well was added with ELISA stop solution (purchased from Solebao), and the absorbance value at 450nm was read.
  • ELISA color developing solution purchased from Soleibo
  • ELISA stop solution purchased from Solebao
  • Example 11 Identification of the crystal structure of the complex between 4-1BB and VHH segment
  • the amino acid sequence of the anti-4-1BB antibody in the final crystallization complex is C in the VHH chain sequence of L-Yr-13 & 14-16-1 (ie SEQ ID NO: 39)
  • a sequence formed by Leu and Gly was added to the end (SEQ ID NO: 169). It should be noted that the Leu and Gly residues added to the C-terminus are only conventional technical operations to promote crystallization, and will not affect Nanobodies L-Yr-13&14-16-1 and human 4-1BB protein binding mode.
  • Crystallographic phase identification was carried out using the molecular replacement method using the structures of 4-1BB (PDB ID: 6mgp) and VHH (PDB ID: 4xt1) as models, respectively. Crystal structure refinement was performed using Refmac5. Leverage COOT for model checking, manual reconstruction and structural verification.
  • the specific crystal data statistics are shown in Table 6.
  • FIG. 9 The crystal structure of the 4-1BB-VHH complex obtained after the structural analysis is shown in FIG. 9 .
  • Epitope analysis showed that the main hydrogen bonds between 4-1BB and VHH were concentrated on amino acids such as Asp118, Leu123, Arg130, Val132, Cys134, Gly135 and Ser137 on 4-1BB ( Figure 10).
  • Leu123, Val124, Val133 and Pro136 on 4-1BB formed hydrophobic interaction interfaces with Val32, Ala33, Tyr37, Leu47, Ile52, Tyr97, Tyr102 and Trp115 on VHH (Fig. 11).
  • the binding site of Urelumab and 4-1BB is located at the N-terminus of 4-1BB.
  • the amino acids that 4-1BB are mainly involved in binding include Pro27, Asn40, Asn42 and Gln43, etc.
  • Utomilumab mainly binds to CRD3 (Cysteine rich domain) and CRD4 of 4-1BB
  • the amino acids that 4-1BB are mainly involved in binding include Arg66, Gly96, Ser100, Cys102, Lys114, Arg130 and Arg134 (Chin SM, Kimberlin CR, Roe-Zurz Z, et al.Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab.

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Abstract

涉及一种靶向4-1BB的单域抗体、其融合蛋白、药物组合物及用途。具体地，涉及的单域抗体包含一个重链可变区，所述重链可变区包含的CDR1-CDR3的氨基酸序列分别如表B中的任一项所示。单域抗体可以结合人的4-1BB，同时交叉结合食蟹猴的4-1BB；借助外部交联的情况下激活T细胞，兼备优异的抗肿瘤活性和安全性。

Description

靶向4-1BB的单域抗体、其融合蛋白、药物组合物及用途 技术领域
本发明属于生物医药领域，涉及一种靶向4-1BB的单域抗体、其融合蛋白、药物组合物及用途。
背景技术
肿瘤坏死因子受体超家族(4-1BB)又名CD137，或TNFRF9，是TNF受体家族的成员之一。4-1BB是一个阅读框含有255个氨基酸(NCBI：NP_001552)的I型跨膜蛋白，由含有17个氨基酸的N端信号肽，169个氨基酸的胞外区，27个氨基酸的跨膜区和42个氨基酸的C端胞内区组成。4-1BB分子主要表达在活化的T细胞、NK细胞、调节性T细胞、树突状细胞、单核细胞、中性粒细胞和嗜酸性细胞中，肿瘤血管的内皮细胞也有报道表达4-1BB。
在T细胞的活化过程中，4-1BB分子能为其提供共刺激信号。当T细胞受体接触到抗原后，会增加4-1BB的表达量，4-1BB与配体结合后会引起NFκB信号通路的激活，从而导致T细胞的活化增殖，并且4-1BB还能抑制活化细胞凋亡。动物模型和体外实验证实，抗4-1BB单抗具有抗肿瘤活性。它能够选择性地引起CD8+T细胞的增殖、上调促炎性细胞因子IFNγ的表达，并且能够增强抗原特异性效应型T细胞的杀伤作用，从而促进肿瘤的清除。抗4-1BB单抗也能引起NK细胞的扩增，并能通过其增加CD8+T细胞的细胞毒活性。抗4-1BB抗体还能够引起肿瘤细胞的血管的内皮细胞上调粘附分子的表达，促进活化的T淋巴细胞浸润肿瘤组织。除此之外，在动物模型中，抗4-1BB激活性抗体还能减轻自身免疫性疾病，如自身免疫性脑脊髓炎，狼疮样综合征和胶原诱导型关节炎。
因此，在肿瘤治疗和一些自身免疫性疾病的治疗中，4-1BB是一个重要潜在的靶点，具体的，在临床前结直肠癌、肺癌、乳腺癌、黑色素瘤等动物模型种，靶向4-1BB的激动剂分子作为单药或者与anti-PD-1、anti-PD-L1、anti-CTLA-4、anti-HER-2等抗体的联用显示出显著的抗肿瘤活性(Etxeberria I,et al.ESMO Open 2020；4:e000733.)。目前有两种4-1BB抗体正在进行临床实验：Urelumab(BMS-663513，Bristol-Myers Squibb开发)和Utomilumab(PF-05082566，Pfizer开发)。研究表明，Urelumab是一种强效的激活剂，具有较好的激活活性，但是也具有肝毒性和疲劳等 不良反应；而Utomilumab在安全性具有优势，但相对于Urelumab，它对受体激动活性较低。
因此，本领域迫切需要开发出一种更高效地靶向4-1BB的抗体及其相关药物。
发明内容
本发明人经过深入的研究和创造性的劳动，开发了一种抗人4-1BB的纳米抗体。具体地，本发明人利用人源的4-1BB抗原蛋白免疫羊驼(Llama)，获得高质量的免疫纳米抗体基因文库。其中，本发明人在所述免疫纳米抗体基因文库中筛选获得可以同时结合人和食蟹猴4-1BB，同时需要借助外部交联发挥激活作用的两株抗4-1BB纳米抗体。再将两株纳米抗体序列进行优化，将基因工程化的突变体表达纯化，从抗体亲和力、与食蟹猴4-1BB交叉结合，激活T细胞的活性等方面进行进一步的筛选，从而获得了一类能在体外高效表达的、且特异性高的纳米抗体株。实验结果表明，本发明的抗4-1BB纳米抗体和融合蛋白具有分子量小，和食蟹猴交叉，不阻断4-1BB配体与4-1BB的天然结合，T细胞激活作用强，安全性好的优点。由此提供了下述发明。
本发明的一个方面涉及一种抗4-1BB单域抗体，其包含一个重链可变区，所述重链可变区包含CDR1-CDR3，其中，CDR1的氨基酸序列选自SEQ ID NOs:41-80，CDR2的氨基酸序列选自SEQ ID NOs:81-120，CDR3的氨基酸序列选自SEQ ID NOs:121-160。
本发明的一个方面涉及一种抗4-1BB单域抗体，其包含一个重链可变区，所述重链可变区包含的CDR1-CDR3的氨基酸序列分别如下面的1-40项中的任一项所示：
Figure PCTCN2021118955-appb-000001
Figure PCTCN2021118955-appb-000002
Figure PCTCN2021118955-appb-000003
本发明中，抗4-1BB单域抗体的CDR均由IMGT编号***定义，请参见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307。
本发明中，所述4-1BB，如果没有特别说明，是指人4-1BB。在本发明的一些实施方式中，所述4-1BB的氨基酸序列如SEQ ID NO:168所示。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其4个框架区中的任意的一个、两个、三个或全部4个进行人源化改造；
优选地，对第二个框架区进行人源化改造；可选地，还对第一个、第三个或第四个框架区进行改造；
优选地，所述抗4-1BB单域抗体与人的同源性大于或等于75％、大于或等于76％、大于或等于77％、大于或等于78％、大于或等于79％、或者大于或等于80％；
优选地，所述抗4-1BB单域抗体的框架区1的氨基酸序列如SEQ ID NO:170所示，框架区2的氨基酸序列如SEQ ID NO:171或SEQ ID NO:172所示，框架区3的氨基酸序列如SEQ ID NO:173所示，并且框架区4的氨基酸序列如SEQ ID NO:174所示。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其与4-1BB抗原的K D为小于E-07、小于5E-08、小于4E-08、小于3E-08、或者小于2E-08；优选地，所述K D通过ForteBio测得。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其与4-1BB抗原的结合位点不同于Urelumbab和Utomilumab。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其特异结合人4-1BB，同时交叉结合食蟹猴4-1BB。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其不阻断天然状态(例如在哺乳动物体内特别是在人体内)下4-1BB配体与4-1BB的结合。
在本发明的一些实施方式中，所述的抗4-1BB单域抗体，其氨基酸序列如SEQ ID NOs:1-40中的任一序列所示。
本发明的另一方面涉及一种融合蛋白，其包含本发明中任一项所述的抗4-1BB单域抗体，以及人IgG的Fc段或人IgG的恒定区。
在本发明的一些实施方式中，所述的融合蛋白，其由本发明中任一项所述的抗4-1BB单域抗体，以及人IgG的Fc段或人IgG的恒定区组成。
在本发明的一些实施方式中，所述的融合蛋白，其由本发明中任一项所述的抗4-1BB单域抗体、连接片段(linker)、以及人IgG的Fc段或人IgG的恒定区组成。
在本发明的一些实施方式中，所述的融合蛋白，其中，按照EU编号***，所述人IgG的Fc段或人IgG的重链恒定区包含L234A突变和L235A突变；可选地，所述IgG的Fc段还包含G237A突变。
在本发明的一些实施方式中，所述融合蛋白为抗4-1BB的重链抗体。重链抗体是指没有轻链的抗体。是目前对VHH-Fc类抗体的一个通用的名称。
在本发明的一些实施方式中，所述的融合蛋白，其中，
所述人IgG的Fc段为人IgG1的Fc段；
所述人IgG的重链恒定区为人IgG1的重链恒定区。
在本发明的一些实施方式中，所述的融合蛋白，其中，
所述人IgG的Fc段为包含L234A突变和L235A突变的人IgG1的Fc段；可选地，所述人IgG1的Fc段还包含G237A突变；
优选地，人IgG1的Fc段的氨基酸序列如SEQ ID NO:167所示。
在本发明的一些实施方式中，所述的融合蛋白，其中，
所述人IgG的Fc段或人IgG的恒定区与所述抗4-1BB单域抗体的C末端直接连接或通过连接片段连接。
在本发明的一些实施方式中，所述的融合蛋白，其中，
所述人IgG1的Fc段或人IgG1的恒定区与所述抗4-1BB单域抗体的C末端直接连接或通过连接片段连接。
本发明的再一方面涉及一种分离的核酸分子，其编码本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白。
本发明还涉及一种载体，其包含本发明的分离的核酸分子。
本发明还涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及一种制备本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗4-1BB单域抗体或融合蛋白的步骤。
本发明的再一方面涉及一种偶联物，其包括抗体部分以及偶联部分，其中，所述抗体部分为本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包含本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白，或者包含本发明的偶联物；
优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述单域抗体或融合蛋白；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白在制备试剂盒中的用途，所述试剂盒用于检测4-1BB在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白或者包含本发明的偶联物；可选地，其还包括药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白或者本发明的偶联物在制备预防和/或治疗恶性肿瘤或自身免疫性疾病的药物中的用途；
优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
本发明的再一方面涉及一种治疗和/或预防恶性肿瘤自身免疫性疾病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗4-1BB单域抗体或者本发明中 任一项所述的融合蛋白或者本发明的偶联物的步骤；
优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
在本发明的一些实施方式中，所述的方法，其中，给予有需求的受试者以有效量的本发明中任一项所述的单域抗体或融合蛋白的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一些实施方式中，所述的方法，其中，
本发明的单域抗体或融合蛋白的单次给药剂量为每千克体重0.1-100mg，优选4.8-24mg或1-10mg；或者，本发明的单域抗体或融合蛋白的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
根据本发明中任一项所述的抗4-1BB单域抗体或者本发明中任一项所述的融合蛋白或者本发明的偶联物，其用于治疗和/或预防恶性肿瘤或自身免疫性疾病；
优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经 典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature1989；342:878-883，或者IMGT编号***定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307的定义。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021118955-appb-000004
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。在一些情况下，抗体的 抗原结合片段是双抗体(Diabodies)，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.et al.,Proc.Natl.Acad.Sci.USA 1993；90:6444-6448和Poljak R.J.et al.,Structure 1994；2:1121-1123)。
如本文中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法(如冷泉港的抗体实验技术指南，5-8章和15章)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“药学上可接受的辅料”是指在药理学和/或生理学上与受 试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19 th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途的有效量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
本发明还涉及如下1至11项中的任意一项：
1.一种抗4-1BB纳米抗体，其特征在于，所述抗4-1BB纳米抗体的VHH链具有如SEQ ID NOs:1-40中任一所示的氨基酸序列；
其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB纳米抗体的4-1BB结合亲和力的衍生序列。
2.一种融合蛋白，其特征在于，所述融合蛋白从N端到C端具有如式I所示的结构：
Z1-L-Z2    (式I)
式中，
Z1为一个或多个(较佳地1-2个，更佳地1个)如第1项所述的抗4-1BB纳米抗体的VHH链；
Z2为免疫球蛋白的Fc段；
L为任选的接头序列。
3.一种多核苷酸，其特征在于，所述多核苷酸编码如第1项所述的抗4-1BB纳米抗体或如第2项所述的融合蛋白。
4.一种表达载体，其特征在于，所述表达载体含有如第3项所述的多核苷酸。
5.一种宿主细胞，其特征在于，所述宿主细胞含有如第4项所述的表达载体，或其基因组中整合有如第3项所述的多核苷酸。
6.一种产生抗4-1BB纳米抗体或融合蛋白的方法，其特征在于，包括步骤：
(a)在适合产生抗4-1BB纳米抗体或融合蛋白的条件下，培养如第5项所述的宿主细胞，从而获得含所述抗4-1BB纳米抗体或融合蛋白的培养物；和
(b)从所述培养物中分离或回收所述的抗4-1BB纳米抗体或融合蛋白。
7.一种免疫偶联物，其特征在于，所述免疫偶联物含有：
(a)如第1项所述的抗4-1BB纳米抗体，或如第2项所述的融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
8.如第1项所述的抗4-1BB纳米抗体或如第2项所述的融合蛋白的用途，用于制备(a)用于检测4-1BB分子的试剂；(b)用于***的药物。
9.一种药物组合物，其特征在于，包括：
(i)如第1项所述的抗4-1BB纳米抗体、如第2项所述的融合蛋白，或如第7项所述的免疫偶联物；和
(ii)药学上可接受的载体。
10.一种重组蛋白，其特征在于，所述的重组蛋白具有：
(i)如第1项所述的抗4-1BB纳米抗体，或如第2项所述的融合蛋白的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
11.一种蛋白复合物，其特征在于，所述的复合物由4-1BB蛋白和抗4-1BB抗体或其抗原结合片段通过氢键和/或疏水相互作用而形成；
其中，所述的氢键作用位点包括4个以上(较佳地5个以上，更佳地6个以上，最佳地为7个)位于4-1BB蛋白中选自下组的位点：第118位Asp、第123位Leu、第130位Arg、第132位Val、第134位Cys、第135位Gly，和第137位Ser；
并且，所述的疏水性相互作用界面的构成位点包括2个以上(较佳地3个以上，更佳地4个以上)位于4-1BB蛋白中选自下组的位点：第123位Leu、第124位Val、第133位Val，和第136位Pro；
其中，所述的4-1BB蛋白的氨基酸编号是基于SEQ ID NO:168的编号。
本发明还涉及如下的第一方面至第十五方面中的任一方面：
在本发明的第一方面，提供了一种抗4-1BB纳米抗体，所述抗4-1BB纳米抗体的VHH链具有如SEQ ID NO:1-40中任一所示的氨基酸序列；
其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB纳米抗体的4-1BB结合亲和力的衍生序列。
在另一优选例中，所述抗4-1BB纳米抗体的VHH链的氨基酸序列如SEQ ID NO:3、35、37、38、39或40中任一所示。
在本发明的第二方面，提供了一种融合蛋白，所述融合蛋白从N端到C端具有如式I所示的结构：
Z1-L-Z2    (式I)
式中，
Z1为一个或多个(较佳地1-2个，更佳地1个)如本发明第一方面所述的抗4-1BB纳米抗体的VHH链；
Z2为免疫球蛋白的Fc段；
L为任选的接头序列。
在另一优选例中，所述的融合蛋白为二聚体，所述的二聚体由Z2中的Fc段之间的二硫键所形成。
在另一优选例中，所述免疫球蛋白是IgG1、IgG2、IgG3或IgG4；优选为IgG1、IgG2或IgG4。
在另一优选例中，所述免疫球蛋白是IgG1，或其突变型。
在另一优选例中，所述L具有选自下组的氨基酸序列：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4、(GGGGS) 5，或其组合。
在另一优选例中，所述Z2的氨基酸序列如SEQ ID NO:167所示。
在另一优选例中，所述Z2的氨基酸序列与如SEQ ID NO:167所示的氨基酸序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个，最佳地为1-3个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加。
在另一优选例中，所述的基本相同是氨基酸序列与相应氨基酸序列的序列同一性至少为70％、至少为75％、至少为80％、至少为85％、至少为86％、至少为87％、 至少为88％、至少为89％、至少为90％、至少为91％、至少为92％、至少为93％、至少为94％、至少为95％、至少为96％、至少为97％、至少为98％，或者至少为99％。
在另一优选例中，所述融合蛋白的式I中，Z1的氨基酸序列如SEQ ID NO:1-40中任一所示，并且L为无，并且Z2的氨基酸序列如SEQ ID NO:167所示；
其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB结合亲和力的衍生序列。
在另一优选例中，所述融合蛋白的式I中，Z1的氨基酸序列如SEQ ID NO:3、35、37、38、39或40中任一所示，并且L为无，并且Z2的氨基酸序列如SEQ ID NO:167所示；
其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB结合亲和力的衍生序列。
在另一优选例中，所述融合蛋白的氨基酸序列中，从N端到C端依次为：如SEQ ID NO:1-40中任一所示的序列和如SEQ ID NO:167所示的序列。
在另一优选例中，所述融合蛋白的氨基酸序列中，从N端到C端依次为：如SEQ ID NO:3、35、37、38、39或40中任一所示的序列，和如SEQ ID NO:167所示的序列。
在本发明的第三方面，提供了一种多核苷酸，所述多核苷酸编码如本发明第一方面所述的抗4-1BB纳米抗体或如本发明第二方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
在本发明的第四方面，提供了一种表达载体，所述表达载体含有如本发明第三方面所述的多核苷酸。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第四方面所述的表达载体，或其基因组中整合有如本发明第三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
在本发明的第六方面，提供了一种产生抗4-1BB纳米抗体或融合蛋白的方法，包括步骤：
(a)在适合产生抗4-1BB纳米抗体或融合蛋白的条件下，培养如本发明第五方面所述的宿主细胞，从而获得含所述抗4-1BB纳米抗体或融合蛋白的培养物；和
(b)从所述培养物中分离或回收所述的抗4-1BB纳米抗体或融合蛋白。
在本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第一方面所述的抗4-1BB纳米抗体，或如本发明第二方面所述的融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2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))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的抗4-1BB纳米抗体，或如本发明第二方面所述的融合蛋白。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的抗4-1BB纳米抗体，或如本发明第二方面所述的融合蛋白。
在本发明的第八方面，提供了如本发明第一方面所述的抗4-1BB纳米抗体或如本发明第二方面所述的融合蛋白的用途，用于制备(a)用于检测4-1BB分子的试剂；(b)用于***的药物。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
在本发明的第九方面，提供了一种药物组合物，包括：
(i)如本发明第一方面所述的抗4-1BB纳米抗体、如本发明第二方面所述的融合蛋白，或如本发明第七方面所述的免疫偶联物；和
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备***的药物，所述的肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、***、淋巴癌、肾上腺肿瘤、***、黑色素瘤、头颈癌、鼻咽癌、甲状腺肿瘤、舌癌或其组合。
在本发明的第十方面，提供了如本发明第一方面所述的抗4-1BB纳米抗体或如本发明第二方面所述的融合蛋白的一种或多种的用途：
(i)用于检测人4-1BB分子；
(ii)用于流式检测；
(iii)用于细胞免疫荧光检测；
(iv)用于***；和
(v)用于肿瘤诊断。
在另一优选例中，所述用途为非诊断的和非治疗的。
在本发明的第十一方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的抗4-1BB纳米抗体，或如本发明第二方面所述的融合蛋白的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括：6His标签、HA标签、Flag标签、Fc标签，或其组合。
在另一优选例中，所述的重组蛋白特异性结合于4-1BB蛋白。
在本发明的第十二方面，提供了如本发明第一方面所述的抗4-1BB纳米抗体、如本发明第二方面所述的融合蛋白，或如本发明第七方面所述的免疫偶联物的用途，被用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中4-1BB蛋白；
其中，所述药剂用于治疗或预防各种血液肿瘤与实体肿瘤。
在另一优选例中，所述肿瘤包括：胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、肾上腺肿瘤、黑色素瘤、头颈癌、鼻咽癌、甲状腺肿瘤、舌癌或其组合。
在本发明的第十三方面，提供了一种检测样品中4-1BB蛋白的方法，包括步骤：
(1)将样品与如本发明第一方面所述的抗4-1BB纳米抗体或如本发明第二方面所述的融合蛋白接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在4-1BB蛋白。
在另一优选例中，所述检测包括定性检测和定量检测。
在本发明的第十四方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用如本发明第一方面所述的抗4-1BB纳米抗体、如本发明第二方面所述的融合蛋白或如本发明第七方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物。
在另一优选例中，所述哺乳动物为人。
在本发明的第十五方面，提供了一种蛋白复合物，所述的复合物由4-1BB蛋白和抗4-1BB抗体或其抗原结合片段通过相互作用而形成。
在另一优选例中，所述的相互作用包括氢键作用和疏水性作用。
在另一优选例中，所述抗4-1BB抗体是抗4-1BB纳米抗体。
在另一优选例中，所述抗4-1BB抗体是如本发明第一方面所述的抗4-1BB纳米抗体。
在另一优选例中，所述的复合物由4-1BB蛋白和抗4-1BB抗体或其抗原结合片段通过氢键和/或疏水相互作用而形成；
其中，所述的氢键作用位点包括4个以上(较佳地5个以上，更佳地6个以上，最佳地7个)位于4-1BB蛋白中选自下组的位点：第118位Asp、第123位Leu、第130位Arg、第132位Val、第134位Cys、第135位Gly，和第137位Ser；
并且，所述的疏水性相互作用界面的构成位点包括2个以上(较佳地3个以上，更佳地4个以上)位于4-1BB蛋白中选自下组的位点：第123位Leu、第124位Val、第133位Val，和第136位Pro；
其中，所述的4-1BB蛋白的氨基酸编号是基于SEQ ID NO:168的编号。
在另一优选例中，所述的疏水性相互作用界面还包括4个以上(较佳地5个以上，更佳地6个以上，最佳地7个以上或8个以上)位于抗4-1BB抗体或其抗原结合片段中选自下组的位点：第32位Val、第33位Ala、第37位Tyr、第47位Leu、第52位Ile、第97位Tyr、第102位Tyr，和第115位Trp；
其中，所述的抗4-1BB抗体或其抗原结合片段的氨基酸编号是基于SEQ ID NO:169或39的编号。
在另一优选例中，所述复合物中的4-1BB蛋白的氨基酸序列如SEQ ID NO:168 所示。
在另一优选例中，所述复合物中的抗4-1BB纳米抗体的氨基酸序列如SEQ ID NO:169或39所示。
在另一优选例中，所述的氢键作用位于4-1BB蛋白的第118位Asp、第123位Leu、第130位Arg、第132位Val、第134位Cys、第135位Gly，和/或第137位Ser；
其中，所述的氨基酸编号是基于SEQ ID NO:168的编号。
在另一优选例中，所述的疏水性作用的作用界面位于4-1BB蛋白的第123位Leu、第124位Val、第133位Val和/或第136位Pro，与抗4-1BB纳米抗体的VHH链的第32位Val、第33位Ala、第37位Tyr、第47位Leu、第52位Ile、第97位Tyr、第102位Tyr和/或第115位Trp；
其中，所述的4-1BB蛋白的氨基酸编号是基于SEQ ID NO:168的编号，并且所述的抗4-1BB纳米抗体的VHH链的氨基酸编号是基于SEQ ID NO:169或39的编号。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
如本文所用，术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
如本文所用，术语“给予”、“施用”可互换使用，是指使用本领域技术人员已知的各种方法和递送***中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
本发明纳米抗体
如本文所用，术语“本发明纳米抗体”、“本发明的抗4-1BB纳米抗体”、“本发明4-1BB纳米抗体”可互换使用，均指特异性识别和结合于4-1BB(包括人4-1BB)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO:1-40中任一所示的纳米抗体，更加优选地是VHH链的氨基酸序列如SEQ ID NO:3、35、37、38、39或40中任一所示的纳米抗体。
如本文所用，术语“单域抗体(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单域抗体(VHH)，它是具 有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
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“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。本发明还包括与所述的抗4-1BB蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“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4-1BB蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。纳米抗体也有4个框架区(framwork)，其中第1、3、4个框架区(框架区1、框架区3和框架区4)和人抗体同源性较高，在人源化过程中改造的必要性较小；优选改造第2个框架区即框架区2(framework 2)。
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4-1BB蛋白结合活性的、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.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))；9.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明融合蛋白
如本文所述，“本发明融合蛋白”是指既具有本发明第一方面所述的抗4-1BB纳米抗体，又具有免疫球蛋白的Fc段的融合蛋白。
在本发明中，提供了一种融合蛋白，所述纳米抗体融合蛋白从N端到C端具有如式I所示的结构：
Z1-L-Z2    (式I)
式中，
Z1为一个或多个如本发明第一方面所述的抗4-1BB纳米抗体的VHH链；
Z2为免疫球蛋白的Fc段；
L为任选的接头序列。
优选地，所述的免疫球蛋白可以是IgG1、IgG2、IgG3或IgG4等。(优选IgG1、IgG2或IgG4)，也可以是IgG1的不同突变型。
在一个实施方式中，所述的融合蛋白为二聚体，所述的二聚体由Z2中的Fc段之间的二硫键所形成。
在一个实施方式中，所述L具有选自下组的氨基酸序列：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4、(GGGGS) 5，或其组合。
在优选的实施方式中，所述Z2的氨基酸序列如SEQ ID NO:167所示。
在另一个实施方式中，所述Z2的氨基酸序列与如SEQ ID NO:167所示的氨基酸序列相同或基本相同。
优选地，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个，最佳地为1-3个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加。
优选地，所述的基本相同是氨基酸序列与相应氨基酸序列的序列同一性至少为 70％、至少为75％、至少为80％、至少为85％、至少为86％、至少为87％、至少为88％、至少为89％、至少为90％、至少为91％、至少为92％、至少为93％、至少为94％、至少为95％、至少为96％、至少为97％、至少为98％，或者至少为99％。
在本发明优选的实施方式中，所述融合蛋白的氨基酸序列中，从N端到C端依次为：如SEQ ID NO:1-40中任一所示的序列和如SEQ ID NO:167所示的序列；
其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB结合亲和力的衍生序列。
本发明的融合蛋白具有双靶标结合亲和力高、特异性强的优势，从而进一步增强抗肿瘤免疫功能。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合4-1BB蛋白分子，因而可用于***。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体 重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的纳米抗体
在本发明的一个优选例中，所述纳米抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗4-1BB的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
本发明的抗4-1BB纳米抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测4-1BB蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中4-1BB蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或融合蛋白或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测4-1BB水平的检测试剂盒，该试剂盒包括识别4-1BB蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的纳米抗体有广泛生物应用价值和临床应用价值，其应用涉及到与4-1BB相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对4-1BB的临床诊断和靶向治疗。
发明的有益效果
本发明取得了如下技术效果中的任意一项或任意多项：
1)本发明的纳米抗体分子量小。
2)本发明的纳米抗体能够高度特异结合人4-1BB蛋白，同时交叉结合食蟹猴蛋白。
3)本发明纳米抗体需要借助外部交联发挥激活作用，具有优异的活性和安全性。
4)本发明的纳米抗体不阻断天然状态下4-1BB配体与4-1BB的结合。
5)作为抗4-1BB的激动型分子，Urelumab因为激活活性太强导致临床肝毒性。本发明的抗体一个重要优势是与Urelumbab和Utomilumab的结合4-1BB的位点有差异，且激活活性处于Urelumab和Utomilumab之间。
附图说明
图1A：本发明第一批的4-1BB抗体结合稳定表达人4-1BB的CHO细胞的检测结果。
图1B：本发明第二批的4-1BB抗体结合稳定表达人4-1BB的CHO细胞的检测结果。
图2A：本发明第一批的4-1BB抗体结合食蟹猴4-1BB蛋白的检测结果。
图2B：本发明第二批的4-1BB抗体结合食蟹猴4-1BB蛋白的检测结果。
图3A：本发明第一批的4-1BB抗体激活原代T细胞产生IFN-γ的检测结果。
图3B：本发明第二批4-1BB抗体激活原代T细胞产生IFN-γ的检测结果。
图4A：本发明L-Yr-13&14-16分子经基因工程改造的子代分子结合稳定表达人4-1BB的CHO细胞的检测结果。
图4B：本发明A-Na-19分子经基因工程改造的子代分子结合稳定表达人4-1BB的CHO细胞的检测结果。
图5A：本发明L-Yr-13&14-16分子经基因工程改造的子代分子结合稳定表达食蟹猴4-1BB的CHO细胞的检测结果。
图5B：本发明A-Na-19分子经基因工程改造的子代分子结合稳定表达食蟹猴4-1BB的CHO细胞的检测结果。
图6A：本发明L-Yr-13&14-16分子经基因工程改造的子代分子激活原代T细胞产生IFN-γ的检测结果。
图6B：本发明A-Na-19分子经基因工程改造的子代分子激活原代T细胞产生IFN-γ的检测结果。
图7A：本发明L-Yr-13&14-16分子经基因工程改造的子代分子阻断4-1BB配体与4-1BB结合的检测结果。
图7B：本发明L-Yr-13&14-16-1分子与TNFRSF成员结合的检测结果。
图8：本发明4-1BB-VHH复合物的晶体示意图。
图9：本发明4-1BB-VHH复合物晶体结构。其中，绿色为4-1BB蛋白；蓝色为抗4-1BB纳米抗体的VHH链。
图10：本发明4-1BB-VHH复合物晶体结构中的氢键相互作用界面。其中，绿色为4-1BB蛋白；蓝色为抗4-1BB纳米抗体的VHH链。
图11：本发明4-1BB-VHH复合物晶体结构中的疏水性相互作用界面。其中，绿色为4-1BB蛋白；蓝色为抗4-1BB纳米抗体的VHH链。
在本发明中，涉及到的抗4-1BB纳米抗体以及其CDR的序列编号如下面的表B所示。
表B：本发明的纳米抗体所对应的序列编号(SEQ ID NO:)
抗体名称 VHH CDR1 CDR2 CDR3
A-Na-16 1 41 81 121
A-Na-18 2 42 82 122
A-Na-19 3 43 83 123
A-Ye-02 4 44 84 124
A-Ye-03 5 45 85 125
A-Ye-04 6 46 86 126
A-Ye-05 7 47 87 127
A-Ye-10 8 48 88 128
A-Ye-13 9 49 89 129
A-Ye-14 10 50 90 130
A-Ye-15 11 51 91 131
A-Ye-18 12 52 92 132
A-Ye-19 13 53 93 133
A-Ye-21 14 54 94 134
A-Ye-22 15 55 95 135
A-Ye-23 16 56 96 136
A-Ye-25 17 57 97 137
A-Ye-27 18 58 98 138
A-Ye-30 19 59 99 139
A-Ye-33 20 60 100 140
A-Ye-20 21 61 101 141
A-Ye-35 22 62 102 142
L-Yr-13&14-01 23 63 103 143
L-Yr-13&14-02 24 64 104 144
L-Yr-13&14-03 25 65 105 145
L-Yr-13&14-05 26 66 106 146
L-Yr-13&14-06 27 67 107 147
L-Yr-13&14-07 28 68 108 148
L-Yr-13&14-08 29 69 109 149
L-Yr-13&14-09 30 70 110 150
L-Yr-13&14-10 31 71 111 151
L-Yr-13&14-11 32 72 112 152
L-Yr-13&14-12 33 73 113 153
L-Yr-13&14-13 34 74 114 154
L-Yr-13&14-16 35 75 115 155
L-Yr-13&14-17 36 76 116 156
Hu-A-NA-19-1 37 77 117 157
Hu-A-NA-19-2 38 78 118 158
HZ-L-Yr-13&14-16-1 39 79 119 159
HZ-L-Yr-13&14-16-2 40 80 120 160
其中，各纳米抗体的CDR均是由IMGT编号***定义。
表C：本发明涉及的部分序列
Figure PCTCN2021118955-appb-000005
Figure PCTCN2021118955-appb-000006
Figure PCTCN2021118955-appb-000007
Figure PCTCN2021118955-appb-000008
Figure PCTCN2021118955-appb-000009
Figure PCTCN2021118955-appb-000010
Figure PCTCN2021118955-appb-000011
Figure PCTCN2021118955-appb-000012
Figure PCTCN2021118955-appb-000013
Figure PCTCN2021118955-appb-000014
Figure PCTCN2021118955-appb-000015
Figure PCTCN2021118955-appb-000016
备注：表C中，SEQ ID NO:41-160为CDR序列，其中部分序列可能相同，为了便于表述，采用了不同的编号。
具体实施方式
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：纳米抗体文库的构建
1.1动物免疫
将1mg人4-1BB抗原(购自AcroBiosystems)与弗氏佐剂等体积混合，免疫2只羊驼(Llama)，每周一次，共免疫4次，剌激B细胞表达抗原特异性的纳米抗体。4次免疫结束后，提取50ml有羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼总cDNA。
1.2纳米抗体基因的扩增
第一轮PCR，从cDNA中扩增出IgG2、IgG3序列：
表1：第一轮PCR引物
名称 序列(5’到3’) SEQ ID NO:
上游引物 GTCCTGGCTGCTCTTCTACAAGG 161
下游引物 GGTACGTGCTGTTGAACTGTTCC 162
将第一轮PCR产物进行琼脂糖凝胶电泳，切胶回收750bp处的片段用于第二轮 VHH序列的扩增。第二轮PCR扩增引物如下：
表2：第二轮PCR引物
Figure PCTCN2021118955-appb-000017
以第二轮PCR产物为模板，进行第三轮PCR，为VHH基因加上同源臂，第三轮PCR扩增引物如下：
表3：第三轮PCR引物
Figure PCTCN2021118955-appb-000018
利用PCR纯化试剂盒(购自QIAGEN)回收目的片段。
1.3文库构建
将线性化的酵母展示载体和第三轮的PCR产物混合后电转化入酿酒酵母(
Figure PCTCN2021118955-appb-000019
20828)中，分别构建来自2只羊驼的抗4-1BB纳米抗体文库。
实施例2：4-1BB纳米抗体的筛选
2.1人4-1BB蛋白的生物素化标记
取适量体积的双蒸水溶解人4-1BB蛋白(购自AcroBiosystems)，按照生物素标记试剂盒(购自Thermo)产品说明书，将生物素溶解后与蛋白溶液混合，于4℃孵育2小时。用脱盐柱(购自Thermo)去除多余的生物素，脱盐柱预处理及样品收集操作均参考产品说明书步骤进行。
2.2 MACS(Magnetic-Activated Cell Sorting)富集能与4-1BB特异性结合的酵母
将实施例1中构建的VHH文库接种于SD-CAA扩增培养基(1L SD-CAA扩增培 养基中含6.7g YNB、5g酪蛋白氨基酸、13.62g Na 2HPO 4·12H 2O、7.44g NaH 2PO 4和2％葡萄糖)中，30℃，225rpm培养过夜。取适量的酵母细胞，3000rpm×5min离心去除培养基，用SD-CAA诱导培养基重悬酵母细胞，诱导过夜。测定诱导后的文库浓度，取适量的酵母细胞，离心去除培养基。用50ml洗涤缓冲液(PBS+0.5％BSA+2mM EDTA)重悬酵母细胞，离心去除上清。用10ml洗涤缓冲液重悬酵母细胞。
加入生物素标记的4-1BB蛋白(终浓度100mM)，室温孵育30min，离心收集酵母细胞，并用50ml洗涤缓冲液洗涤酵母3遍。用5ml洗涤缓冲液重悬酵母细胞，并加入200μl SA磁珠(购自美天旎)，颠倒孵育10min。用洗涤缓冲液洗涤酵母和磁珠混合物3遍，将混合物加入LS柱(购自美天旎)中。将LS柱放在磁力架上，用洗涤缓冲液洗涤去除非特异性结合的酵母细胞。将柱子从磁力架上取出，加入洗涤缓冲液洗脱酵母。洗脱下来的酵母离心后转入200ml SD-CAA扩增培养基中进行扩增。
2.3流式细胞分选获得高亲和力酵母细胞
将经过MACS富集的酵母细胞接种于SD-CAA扩增培养基中，30℃，225rpm摇瓶培养过夜。用SD-CAA诱导培养基(1L SD-CAA诱导培养基中含6.7g YNB，5g酪蛋白氨基酸，13.62g Na 2HPO 4·12H 2O，7.44g NaH 2PO 4及2％半乳糖，2％棉子糖和0.1％葡萄糖)重悬酵母细胞，诱导过夜。加入1:200稀释的anti-c-Myc鼠源抗体(购自Thermo)和100nM生物素标记的4-1BB抗原，室温孵育10min。加入PBS清洗酵母3遍，加入1:500稀释的羊抗鼠IgG(H+L)Alexa Fluor Plus 488(购自Invitrogen)和链霉亲和素APC偶联荧光抗体(购自Invitrogen)，4℃避光孵育15min。加入2ml PBS重悬细胞，使用BD FACS AriaII仪器进行分选获得可与4-1BB抗原有较高结合能力的酵母。
2.4 4-1BB纳米抗体候选分子抗体基因的获取
通过MACS和FACS富集得到的能与4-1BB抗原有较高结合能力的酵母菌液，在SD-CAA扩增培养基中30℃，225rpm培养过夜，按照酵母质粒抽提试剂盒(购自天根)操作抽提酵母质粒。质粒通过电转化入Top10感受态细胞(购自天根)，涂布氨苄抗性平板，于37℃培养过夜。挑取单克隆测序获得VHH基因序列。
实施例3：重链抗体融合蛋白的构建及表达纯化
3.1抗体基因构建入pCDNA3.1表达载体
将实施例2获得的VHH基因序列和人IgG1(LALA突变)Fc段相连，利用同源重 组酶(购自Vazyme)和EcoR I/Not I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序。
3.2细胞转染
采用ExpiCHO TM表达***试剂盒(购自Thermo)，将质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自南京金斯瑞生物科技有限公司)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自苏州海狸生物医学工程有限公司)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH 3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀。纯化后得到的4-1BB抗体融合蛋白样品用于下面的实施例4至实施例7。
实施例4：4-1BB抗体融合蛋白亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于溶液的高通量抗体-抗原亲和力和表位分级的测量，MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的4-1BB抗原中作用5min，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
实验结果如下面的表4所示。
表4：候选分子亲和力
Figure PCTCN2021118955-appb-000020
Figure PCTCN2021118955-appb-000021
Figure PCTCN2021118955-appb-000022
结果显示，本发明的抗4-1BB分子单价亲和力在1E-08水平，符合激动型分子的开发要求。
实施例5：4-1BB抗体融合蛋白与人4-1BB CHO-S细胞结合
通过转染克隆到MCS(多克隆位点)的编码人4-1BB cDNA的pCHO1.0载体(购自Invitrogen)产生过表达人4-1BB的CHO细胞(CHO-h4-1BB细胞)。将扩大培养的CHO-h4-1BB细胞调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离心备用。将纯化的4-1BB抗体用PBS稀释，400nM开始3倍稀释共12个点，将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
实验结果如图1A、图1B所示。
结果显示，本发明的所有纯化样品均具有与CHO-h4-1BB细胞结合的活性，且部分纯化样品的活性与对照抗体Urelumab(US20090068192)相似。
实施例6：4-1BB抗体融合蛋白与食蟹猴4-1BB蛋白结合
食蟹猴4-1BB-his蛋白(购自ACRO)按照说明书溶解，用ELISA包被液(购自上海生工)稀释至1μg/ml，100μl/孔包被ELISA板，4℃过夜，PBST清洗3次，5％BSA(购自上海生工)室温封闭1小时。弃去包被液，100μl/孔加入1％BSA梯度稀释的4-1BB抗体，200nM 3倍稀释共12个点，室温孵育2小时。PBST清洗3次，100μl/孔加入1％BSA稀释的羊抗人IgG-Fc-HRP(购自abcam)，室温孵育1小时。PBST清洗3次，100μl/孔加入ELISA显色液(购自索莱宝)室温反应3分钟，50μl/孔加入ELISA终止液(购自索莱宝)，读取450nm处吸光度数值。
实验结果如图2A、图2B所示，本发明的部分纯化样品(例如L-Yr-13&14-16和A-Na-19)和对照抗体Utomilumab与食蟹猴4-1BB蛋白有较好的结合活性，对照抗体Urelumab(US20090068192)与食蟹猴4-1BB蛋白结合活性较弱。
实施例7：4-1BB抗体融合蛋白激活原代T细胞的测定
OKT-3抗体(购自Biolegend)用无菌PBS(购自Hyclone)稀释至1μg/ml，50μl/孔包被96孔细胞培养平底板(购自Thermo)，同时50μl/孔加入PBS梯度稀释的抗4-1BB抗体，37℃孵育两小时。复苏冻存的人PBMC(购自上海赛笠)，用人T细胞分离纯化试剂盒(购自Stemcell)分离出T细胞，用X-VIVO15培养基(购自LONZA)重悬T细胞，调整细胞密度为0.5×10 6细胞/ml，备用。待抗体包被完成，弃去包被液，PBS清洗两次，弃去PBS，200μl/孔加入上述T细胞悬液。37℃，5％CO 2培养5天，收集上清检测IFN-γ含量。
实验结果如图3A、图3B所示，本发明的部分纯化样品可以激活人原代T细胞分泌IFN-γ，且部分样品(例如A-Na-16、A-Na-18、A-Na-19)的活性和对照抗体Urelumab(US20090068192)相当，部分样品(例如L-Yr-13&14-16、L-Yr-13&14-17)的活性处于Urelumab和Utomiumab之间。
实施例8：4-1BB抗体的人源化改造和表达
为了减少单克隆抗体在人体内产生免疫原性，将L-Yr-13&14-16和A-Na-19抗体进行人源化。人源化方法采用VHH人源化通用框架移植法，同时根据文献(Vincke,C.,等人，人源化骆驼单域抗体和鉴定通用人源化纳米抗体支架的一般策略.J Biol Chem 284(5):3273-3284)报道的方法对抗体框架2(framework 2)的部分氨基酸进行突变完成。分别得到序列SEQ ID NOs:39-40，以及SEQ ID NOs:37-38。
本研究利用IMGT(http://www.imgt.org)对L-Yr-13&14-16和A-Na-19抗体人源化序列进行了人源化水平评估，结果如表5所示。
表5：L-Yr-13&14-16和A-Na-19人源化序列和人的同源性(比较VHH的序列)
编号 种系 同源性 SEQ ID NO:
L-Yr-13&14-16 IGHV3-30*01 76.3％ 35
HZ-L-Yr-13&14-16-1 IGHV3-30*01 80.4％ 39
HZ-L-Yr-13&14-16-2 IGHV3-30*01 84.5％ 40
A-Na-19 IGHV3-30*01 69.4％ 3
Hu-A-NA-19-1 IGHV3-15*06 76.5％ 37
Hu-A-NA-19-2 IGHV3-15*06 78.6％ 38
蛋白构建及表达纯化方法同实施例3。对纯化的基因工程改造的抗体进行人4-1BB  CHO-S细胞结合检测，检测方法同实施例4。结果如图4A、图4B所示，基因工程改造后的抗体保持与人4-1BB CHO-S细胞结合活性。
对纯化的基因工程改造的抗体进行食蟹猴4-1BB CHO-S细胞结合检测。结果如图5A、图5B所示。结果显示，基因工程改造后的HZ-L-Yr-13&14-16抗体保持与食蟹猴4-1BB CHO-S细胞结合活性，而基因工程改造后的A-Na-19抗体HZ-A-Na-19-1、HZ-A-Na-19-2与食蟹猴4-1BB CHO-S细胞结合活性下降明显。
对纯化的基因工程改造的抗体进行原代T细胞激活测定，检测方法同实施例7。结果如图6A、图6B所示。结果显示，基因工程改造后的HZ-L-Yr-13&14-16-1、HZ-A-Na-19-1保持原代T细胞激活活性。
实施例9：4-1BB配体阻断试验
将扩大培养的CHO-h4-1BB细胞调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离心备用。将纯化的4-1BB抗体用PBS稀释至400nM，将上述稀释好的样品50μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min。50μl/孔加入PBS稀释至1μg/ml的Biotin-4-1BB ligand蛋白(购自ACRO)，4℃孵育30min。PBS清洗两次，100μl/孔加入用PBS稀释200倍的SAPE(购自Thermo)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
结果如图7A所示。结果显示，基因工程改造的4-1BB抗体HZ-L-Yr-13&14-16-1无配体阻断活性，对照抗体Utomilumab(US20120237498)有配体阻断活性，而Urelumab有部分阻断活性。
实施例10：抗体和TNFRSF成员结合检测
人4-1BB-his、GITR-his、OX40-his、CD40-his蛋白(购自ACRO)按照说明书溶解，用ELISA包被液(购自上海生工)稀释至1μg/ml，100μl/孔包被ELISA板，4℃过夜，PBST清洗3次，200μl/孔加入5％BSA(购自上海生工)室温封闭1小时。弃去包被液，100μl/孔加入1％BSA稀释的4-1BB抗体，200nM 3个复孔，室温孵育2小时。PBST清洗3次，100μl/孔加入1％BSA稀释的羊抗人IgG-Fc-HRP(购自abcam)，室温孵育1小时。PBST清洗3次，100μl/孔加入ELISA显色液(购自索莱宝)室温反应3分钟，50μl/孔加入ELISA终止液(购自索莱宝)，读取450nm处吸光度数值。
结果如图7B所示。结果显示，HZ-L-Yr-13&14-16-1抗体仅和4-1BB蛋白结合，和其它肿瘤坏死因子受体超家族成员不结合。说明本发明抗体的特异性好，不会再体内激活其它肿瘤坏死因子受体超家族成员，避免了潜在的副作用。
实施例11：4-1BB与VHH段复合物晶体结构鉴定
为进一步探究本发明的4-1BB纳米抗体与其抗原蛋白的结合模式，在本实施例中，选取L-Yr-13&14-16-1，进行了抗原复合物的结晶实验。
其中，为促进晶体的形成，经优化，最终结晶复合物中的抗4-1BB抗体的氨基酸序列是在L-Yr-13&14-16-1的VHH链序列(即SEQ ID NO:39)的C端增加了Leu和Gly所形成的序列(SEQ ID NO:169)。需要特别说明的是，该C端所添加的Leu和Gly残基仅为促进结晶所进行的常规技术操作，而并不会影响纳米抗体L-Yr-13&14-16-1与人源4-1BB蛋白的结合模式。
本实验采用X光衍射法鉴定4-1BB与VHH段复合物晶体结构。人源4-1BB蛋白(SEQ ID NO:168)与抗4-1BB VHH蛋白(SEQ ID NO:169)均由HEK293体系表达。4-1BB与VHH以摩尔比1:1比例混合制备用于结晶的复合物样品。复合物(8.5mg/mL)与结晶试剂以1:1比例混合并在18℃下进行晶体培养。四天后在JBK试剂盒培养条件中观察到晶体，晶体形貌如图8所示。
挑选单晶在上海光源进行X光衍射实验并收获分辨率为
Figure PCTCN2021118955-appb-000023
的衍射数据。数据处理使用XDS软件。利用分子置换法分别以4-1BB(PDB ID:6mgp)和VHH(PDB ID:4xt1)结构为模型进行晶相鉴定。利用Refmac5进行晶体结构精修。利用COOT进行模型检测、手动重建和结构校验。该复合物晶体归属于P41212空间群，其晶胞参数分别为：
Figure PCTCN2021118955-appb-000024
α＝90.00°，β＝90.00°，γ＝90.00°。具体晶体数据统计见表6。
表6 4-1BB-VHH复合物晶体数据统计
Figure PCTCN2021118955-appb-000025
Figure PCTCN2021118955-appb-000026
结构解析后所得4-1BB-VHH复合物晶体结构如图9所示。表位分析可知，4-1BB与VHH间主要氢键作用集中于4-1BB上的Asp118、Leu123、Arg130、Val132、Cys134、Gly135和Ser137等氨基酸(图10)。此外，4-1BB上Leu123、Val124、Val133和Pro136与VHH上Val32、Ala33、Tyr37、Leu47、Ile52、Tyr97、Tyr102和Trp115构成了疏水性作用界面(图11)。
Urelumab与4-1BB结合位点位于4-1BB的N端，4-1BB主要参与结合的氨基酸包括Pro27、Asn40、Asn42和Gln43等；Utomilumab主要结合在4-1BB的CRD3(Cysteine rich domain)和CRD4，4-1BB主要参与结合的氨基酸包括Arg66、Gly96、Ser100、Cys102、Lys114、Arg130和Arg134(Chin SM,Kimberlin CR,Roe-Zurz Z,et al.Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab.Nat Commun 2018；9:4679.)。另外，从理论上，Utomilumab结合表位与4-1BB配体在空间上存在竞争关系，故会对4-1BB与其配体的结合具有一定阻断作用。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (34)

  1. 抗4-1BB单域抗体，包含一个重链可变区，所述重链可变区包含CDR1-CDR3，其中，CDR1的氨基酸序列选自SEQ ID NOs:41-80，CDR2的氨基酸序列选自SEQ ID NOs:81-120，CDR3的氨基酸序列选自SEQ ID NOs:121-160；
    优选地，所述CDR1-CDR3的氨基酸序列分别如下面的1-40项中的任一项所示：
    Figure PCTCN2021118955-appb-100001
    Figure PCTCN2021118955-appb-100002
  2. 根据权利要求1所述的抗4-1BB单域抗体，其4个框架区中的任意的一个、两个、三个或全部4个进行人源化改造；
    优选地，对第二个框架区进行人源化改造；可选地，还对第一个、第三个或第四个框架区进行改造；
    优选地，所述抗4-1BB单域抗体与人的同源性大于或等于75％、大于或等于76％、大于或等于77％、大于或等于78％、大于或等于79％、或者大于或等于80％；
    优选地，所述抗4-1BB单域抗体的框架区1的氨基酸序列如SEQ ID NO:170所示，框架区2的氨基酸序列如SEQ ID NO:171或SEQ ID NO:172所示，框架区3的氨基酸序列如SEQ ID NO:173所示，并且框架区4的氨基酸序列如SEQ ID NO:174所示。
  3. 根据权利要求1至2中任一权利要求所述的抗4-1BB单域抗体，其与4-1BB抗原的K D为小于E-07、小于5E-08、小于4E-08、小于3E-08、或者小于2E-08；优选地，所述K D通过ForteBio测得。
  4. 根据权利要求1至3中任一权利要求所述的抗4-1BB单域抗体，其与4-1BB抗原的结合位点不同于Urelumbab和Utomilumab。
  5. 根据权利要求1至4中任一权利要求所述的抗4-1BB单域抗体，其特异结合人4-1BB，同时交叉结合食蟹猴4-1BB。
  6. 根据权利要求1至5中任一权利要求所述的抗4-1BB单域抗体，其不阻断天然状态下4-1BB配体与4-1BB的结合。
  7. 根据权利要求1至6中任一权利要求所述的抗4-1BB单域抗体，其氨基酸序列如SEQ ID NOs:1-40中的任一序列所示。
  8. 一种融合蛋白，其包含权利要求1至7中任一权利要求所述的抗4-1BB单域抗体，以及人IgG的Fc段或人IgG的恒定区。
  9. 根据权利要求8所述的融合蛋白，其中，按照EU编号***，所述人IgG的Fc段或人IgG的重链恒定区包含L234A突变和L235A突变；可选地，所述IgG的Fc段还包含G237A突变。
  10. 根据权利要求8至9中任一权利要求所述的融合蛋白，其中，
    所述人IgG的Fc段为人IgG1的Fc段；
    所述人IgG的重链恒定区为人IgG1的重链恒定区。
  11. 根据权利要求8至10中任一权利要求所述的融合蛋白，其中，
    所述人IgG的Fc段为包含L234A突变和L235A突变的人IgG1的Fc段；可选地， 所述人IgG1的Fc段还包含G237A突变；
    优选地，人IgG1的Fc段的氨基酸序列如SEQ ID NO:167所示。
  12. 根据权利要求8至11中任一权利要求所述的融合蛋白，其中，
    所述人IgG的Fc段或人IgG的恒定区与所述抗4-1BB单域抗体的C末端直接连接或通过连接片段连接。
  13. 分离的核酸分子，其编码权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白。
  14. 一种载体，其包含权利要求13所述的分离的核酸分子。
  15. 一种宿主细胞，其包含权利要求13所述的分离的核酸分子，或者权利要求14所述的载体。
  16. 制备权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白的方法，其包括在合适的条件下培养权利要求15的宿主细胞，以及从细胞培养物中回收所述抗4-1BB单域抗体或融合蛋白的步骤。
  17. 偶联物，其包括抗体部分以及偶联部分，其中，所述抗体部分为权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  18. 试剂盒，其包含权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白，或者包含权利要求17所述的偶联物；
    优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述单域抗体或融合蛋白；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  19. 权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白在制备试剂盒中的用途，所述试剂盒用于检测4-1BB在样品中的存在或其水平。
  20. 一种药物组合物，其包含权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白或者包含权利要求17所述的偶联物；可选地，其还包括药学上可接受的辅料。
  21. 权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白或者权利要求17所述的偶联物在制备预防和/或治疗恶性肿瘤或自身免疫性疾病的药物中的用途；
    优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
    优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
  22. 一种治疗和/或预防恶性肿瘤或自身免疫性疾病的方法，包括给予有需求的受试者以有效量的权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白或者权利要求17所述的偶联物的步骤；
    优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
    优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
  23. 根据权利要求1至7中任一权利要求所述的抗4-1BB单域抗体或者权利要求8至12中任一权利要求所述的融合蛋白或者权利要求17所述的偶联物，其用于治疗和/或预防恶性肿瘤或自身免疫性疾病；
    优选地，所述恶性肿瘤选自直肠癌、结肠癌、肺癌、乳腺癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、食道癌和头颈癌；
    优选地，所述自身免疫性疾病选自自身免疫性脑脊髓炎、狼疮样综合征和胶原诱导型关节炎。
  24. 一种抗4-1BB纳米抗体，其特征在于，所述抗4-1BB纳米抗体的VHH链具有如SEQ ID NOs:1-40中任一所示的氨基酸序列；
    其中，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1-8个(较佳地1-5个，更佳地1-3个)氨基酸残基，并能够保留4-1BB纳米抗体的4-1BB结合亲和力的衍生序列。
  25. 一种融合蛋白，其特征在于，所述融合蛋白从N端到C端具有如式I所示的结构：
    Z1-L-Z2  (式I)
    式中，
    Z1为一个或多个(较佳地1-2个，更佳地1个)如权利要求24所述的抗4-1BB纳米抗体的VHH链；
    Z2为免疫球蛋白的Fc段；
    L为任选的接头序列。
  26. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求24所述的抗4-1BB纳米抗体或如权利要求25所述的融合蛋白。
  27. 一种表达载体，其特征在于，所述表达载体含有如权利要求26所述的多核苷酸。
  28. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求27所述的表达载体，或其基因组中整合有如权利要求26所述的多核苷酸。
  29. 一种产生抗4-1BB纳米抗体或融合蛋白的方法，其特征在于，包括步骤：
    (a)在适合产生抗4-1BB纳米抗体或融合蛋白的条件下，培养如权利要求28所述的宿主细胞，从而获得含所述抗4-1BB纳米抗体或融合蛋白的培养物；和
    (b)从所述培养物中分离或回收所述的抗4-1BB纳米抗体或融合蛋白。
  30. 一种免疫偶联物，其特征在于，所述免疫偶联物含有：
    (a)如权利要求24所述的抗4-1BB纳米抗体，或如权利要求25所述的融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  31. 如权利要求24所述的抗4-1BB纳米抗体或如权利要求25所述的融合蛋白的用途，用于制备(a)用于检测4-1BB分子的试剂；(b)用于***的药物。
  32. 一种药物组合物，其特征在于，包括：
    (i)如权利要求24所述的抗4-1BB纳米抗体、如权利要求25所述的融合蛋白，或如权利要求30所述的免疫偶联物；和
    (ii)药学上可接受的载体。
  33. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求24所述的抗4-1BB纳米抗体，或如权利要求25所述的融合蛋白的序列；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  34. 一种蛋白复合物，其特征在于，所述的复合物由4-1BB蛋白和抗4-1BB抗体或其抗原结合片段通过氢键和/或疏水相互作用而形成；
    其中，所述的氢键作用位点包括4个以上(较佳地5个以上，更佳地6个以上，最佳地为7个)位于4-1BB蛋白中选自下组的位点：第118位Asp、第123位Leu、第130位Arg、第132位Val、第134位Cys、第135位Gly，和第137位Ser；
    并且，所述的疏水性相互作用界面的构成位点包括2个以上(较佳地3个以上，更佳地4个以上)位于4-1BB蛋白中选自下组的位点：第123位Leu、第124位Val、第133位Val，和第136位Pro；
    其中，所述的4-1BB蛋白的氨基酸编号是基于SEQ ID NO:168的编号。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024061223A1 (zh) * 2022-09-20 2024-03-28 普米斯生物技术(珠海)有限公司 抗体及其在抗肿瘤中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117736330B (zh) * 2024-01-24 2024-05-17 上海沙砾生物科技有限公司 肿瘤坏死因子超家族受体9的特异性抗原结合蛋白及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US20090068192A1 (en) 2003-10-10 2009-03-12 Bristol-Myers Squibb Company Fully human antibodies against human 4-1BB
US20120237498A1 (en) 2010-09-09 2012-09-20 Pfizer Inc 4-1bb binding molecules
CN109970865A (zh) * 2017-12-28 2019-07-05 上海细胞治疗研究院 一种cd137双向激活共刺激分子受体及其用途
CN111542342A (zh) * 2017-12-05 2020-08-14 礼进生物医药科技（上海）有限公司 抗cd137抗体及其用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110392696B (zh) * 2017-01-06 2020-09-11 优特力克斯有限公司 抗-人4-1bb抗体及其应用
US20210363266A1 (en) * 2018-01-22 2021-11-25 Jiangsu Hengrui Medicine Co., Ltd. Anti-4-1bb antibody, antigen-binding fragment thereof and medical use thereof
CN112646031B (zh) * 2019-10-10 2021-12-07 天境生物科技(上海)有限公司 抗4-1bb纳米抗体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US20090068192A1 (en) 2003-10-10 2009-03-12 Bristol-Myers Squibb Company Fully human antibodies against human 4-1BB
US20190062445A1 (en) * 2003-10-10 2019-02-28 Bristol-Myers Squibb Company Fully human antibodies against human 4-1bb
US20120237498A1 (en) 2010-09-09 2012-09-20 Pfizer Inc 4-1bb binding molecules
CN105481983A (zh) * 2010-09-09 2016-04-13 辉瑞公司 4-1bb结合分子
CN111542342A (zh) * 2017-12-05 2020-08-14 礼进生物医药科技（上海）有限公司 抗cd137抗体及其用途
CN109970865A (zh) * 2017-12-28 2019-07-05 上海细胞治疗研究院 一种cd137双向激活共刺激分子受体及其用途

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
BAGHERI SALMAN, SAFAIE QAMSARI ELMIRA, YOUSEFI MEHDI, RIAZI-RAD FARHAD, SHARIFZADEH ZAHRA: "Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response", CELLULAR & MOLECULAR BIOLOGY LETTERS, vol. 25, no. 1, 1 December 2020 (2020-12-01), PL , pages 1 - 13, XP055911687, ISSN: 1425-8153, DOI: 10.1186/s11658-020-00219-8 *
BAGHERI, S. ET AL.: "Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.", TUMOR BIOLOGY., vol. 39, no. 3, 31 March 2017 (2017-03-31) *
CHEN ZHE-HAO;LI LIN-HONG;CAO WANG-LI;JIN ZHU-MING;FANG LING: "Characteristics and Clinical Development Status of Therapeutic Single Domain Antibody (sdAb)", CHINESE JOURNAL OF NEW DRUGS, vol. 28, no. 21, 15 December 2019 (2019-12-15), pages 2581 - 2587, XP055911686, ISSN: 1003-3734 *
CHIN SMKIMBERLIN CRROE-ZURZ Z ET AL.: "Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab", NAT COMMUN, vol. 9, 2018, pages 4679
CHOTHIA & LESK J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CLARK, IMMUNOL. TODAY, vol. 21, 2000, pages 397 - 402
EHRENMANN FKAAS QLEFRANC M P.: "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J", NUCLEIC ACIDS RESEARCH, vol. 38, 2009, pages D301 - D307, XP055247165, DOI: 10.1093/nar/gkp946
ESTEP, P ET AL.: "Solution-based measurement of high-throughput antibody-antigen affinity and epitope classification", MABS, vol. 5, no. 2, 2013, pages 270 - 8
HOLLIGER P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993
JONES ET AL., NATURE, 1986, pages 321
KABAT ET AL., NIH, vol. I, no. 91-3242, 1991, pages 647 - 669
KOHLER GMILSTEIN C.: "Continuous cultures of fused cells secreting antibody of predefined specificity [J", NATURE, vol. 256, no. 5517, 1975, pages 495, XP037052082, DOI: 10.1038/256495a0
NEIL H. SEGAL, THEODORE F. LOGAN, F. STEPHEN HODI, DAVID MCDERMOTT, IGNACIO MELERO, OMID HAMID, HENRIK SCHMIDT, CAROLINE ROBERT, V: "Results from an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody", CLINICAL CANCER RESEARCH, vol. 23, no. 8, 15 April 2017 (2017-04-15), US, pages 1929 - 1936, XP055448193, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-16-1272 *
POLJAK R. J. ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 1123
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
REICHMANN ET AL., NATURE, vol. 332, 1988, pages 323329
SAMBROOK ET AL.: "Molecular cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
VINCKE, C. ET AL.: "General strategy for humanizing camelid single-domain antibodies and identifying universal humanized nanobody scaffolds", J BIOL CHEM, vol. 284, no. 5, pages 3273 - 3284

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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