WO2023066336A1 - 抗muc17纳米抗体及其应用 - Google Patents

抗muc17纳米抗体及其应用 Download PDF

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WO2023066336A1
WO2023066336A1 PCT/CN2022/126410 CN2022126410W WO2023066336A1 WO 2023066336 A1 WO2023066336 A1 WO 2023066336A1 CN 2022126410 W CN2022126410 W CN 2022126410W WO 2023066336 A1 WO2023066336 A1 WO 2023066336A1
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antigen
nanobody
mutations
antibody
seq
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PCT/CN2022/126410
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English (en)
French (fr)
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成广存
李玲
付雅媛
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to CN202280069376.0A priority Critical patent/CN118251418A/zh
Publication of WO2023066336A1 publication Critical patent/WO2023066336A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present application relates to the field of antibodies, in particular, to anti-MUC17 Nanobodies.
  • Nanobodies generally have longer CDR3s and can form stable large protrusions.
  • the loop structure can bind some cryptic epitopes, and is especially suitable for targets that are difficult to obtain antibodies, such as GPCRs, ion channels, and enzyme activity centers.
  • VHH nanobodies have a small molecular weight, are easy to express in vitro, have good solubility, and have weak immunogenicity. They can pass through some protective barriers in the body and enter the diseased site to play a role, such as the blood-brain barrier.
  • Mucin is a family of high-molecular-weight glycoproteins, which can be divided into secretory proteins and transmembrane proteins. Its protein backbone rich in serine and threonine is combined with a variety of O-type oligosaccharide side chains. Typical oligosaccharide chains It accounts for about 70% of mature mucin.
  • Membrane-bound mucins are cell surface mucins that rely on the transmembrane region to bind to the cell membrane. Members include MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16 and MUC17.
  • MUC17 (Mucin-17) is a single transmembrane mucin with high homology to MUC3 and was first discovered in 2002.
  • the full length of MUC17 protein is 4493 amino acids, the extracellular part contains 59 tandem repeats rich in serine, threonine and proline, 2 EGF and 1 SEA domain, and the intracellular part contains 80 amino acid cytoplasmic tail
  • the phosphorylation site the phosphorylation modification of the intracellular segment may be related to the signal transduction of cell proliferation and apoptosis.
  • Different types of membrane-bound mucins have different expression sites. For example, in normal humans, MUC1 is mainly expressed in the stomach, and MUC17 is mainly expressed in the intestine. In tumor tissue, MUC17 is highly expressed in both gastric cancer and pancreatic cancer, and belongs to tumor-associated antigens. The development of drugs targeting MUC17 is of great significance to the clinical treatment of gastric cancer and pancreatic cancer.
  • the present application screens and obtains MUC17-specific nanobodies, which are intended to be used in the treatment of different forms of malignant tumors.
  • the present application provides an anti-MUC17 antibody, a nucleic acid encoding the same, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.
  • the application provides a Nanobody or an antigen-binding fragment thereof that specifically binds to MUC17, wherein the Nanobody or an antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are selected from From CDR1, CDR2 and CDR3 of the VHH shown in any one of SEQ ID NO.8 ⁇ 12, 62 ⁇ 66, 67 ⁇ 98.
  • the present application provides a multispecific molecule comprising the Nanobody or antigen-binding fragment thereof of the first aspect.
  • the present application provides a chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain Comprising the Nanobody or antigen-binding fragment thereof of the first aspect.
  • CAR chimeric antigen receptor
  • the present application provides an immune effector cell expressing the chimeric antigen receptor described in the third aspect, or comprising a nucleic acid fragment encoding the chimeric antigen receptor described in the third aspect.
  • the application provides an isolated nucleic acid fragment encoding the Nanobody or antigen-binding fragment thereof of the first aspect, or the multispecific molecule of the second aspect, or the multispecific molecule of the third aspect. chimeric antigen receptors.
  • the present application provides a vector comprising the nucleic acid fragment described in the fifth aspect.
  • the present application provides a host cell comprising the vector described in the sixth aspect.
  • the present application provides a method for preparing the Nanobody or antigen-binding fragment thereof of the first aspect or the multispecific molecule of the second aspect, comprising culturing the cell of the seventh aspect, and isolating Nanobodies or antigen-binding fragments thereof expressed by said cells, or isolating multispecific molecules expressed by said cells.
  • the present application provides a method for preparing the immune effector cell described in the fourth aspect, which includes introducing the nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cell.
  • the present application provides a pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof described in the first aspect, the multispecific antibody described in the second aspect, and the immune effector described in the fourth aspect
  • the present application provides the Nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the immune effector cell of the fourth aspect, and the nucleic acid of the fifth aspect Fragments, the carrier of the sixth aspect, the host cell of the seventh aspect, the product obtained by the method of the eighth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of prevention and/or treatment of tumors Uses in medicine.
  • the present application provides a method for preventing and/or treating tumors, comprising administering an effective amount of the Nanobody or antigen-binding fragment thereof described in the first aspect to a patient in need thereof, and the nanobody or antigen-binding fragment thereof described in the second aspect.
  • the present application provides the Nanobody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the immune effector cell of the fourth aspect, and the nucleic acid of the fifth aspect
  • the present application provides a kit comprising the Nanobody or antigen-binding fragment thereof described in the first aspect, the multispecific antibody described in the second aspect, and the immune effector described in the fourth aspect
  • the present application provides a method for detecting the expression of MUC17, wherein under the condition that a complex can be formed between the Nanobody or its antigen-binding fragment described in the first aspect and MUC17, the sample to be detected is mixed with The Nanobody or antigen-binding fragment thereof of the first aspect is contacted.
  • the present application provides a method for inhibiting the proliferation or migration of cells expressing MUC17 in vitro, wherein the conditions under which a complex can be formed between the Nanobody or its antigen-binding fragment described in the first aspect and MUC17 Next, the cell is contacted with the Nanobody or antigen-binding fragment thereof of the first aspect.
  • Figure 1A shows the ELISA detection of the binding reaction of Lab315-huFc recombinant antibody to human MUC17 ECD4131-his protein.
  • Figure 1B shows the ELISA detection of the binding reaction of Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc recombinant antibodies to human MUC17 ECD4131-his protein.
  • Figure 2A is ELISA detection of the binding reaction of Lab315-huFc recombinant antibody to monkey MUC17 ECD3577-His protein.
  • Figure 2B is ELISA detection of the binding reaction of Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc recombinant antibodies to monkey MUC17 ECD3577-His protein.
  • Fig. 3A is the FACS detection of the binding reaction of Lab315-huFc recombinant antibody to NUGC4 endogenous tumor cells.
  • Fig. 3B is the FACS detection of the binding reaction of Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc recombinant antibodies to NUGC4 endogenous tumor cells.
  • Fig. 4A is the FACS detection of the binding of the Lab315-huFc recombinant antibody to the monkey MUC17 overexpression cell FlpinCHO-Cyno(3597-3964)-D2.
  • Fig. 4B is the FACS detection of the binding of Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc recombinant antibodies to the monkey MUC17 overexpression cell FlpinCHO-Cyno(3597-3964)-D2.
  • Figures 5A-5E are the ELISA detection of the binding reactions of SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 humanized antibodies to human MUC17 ECD4131-his protein, respectively.
  • Figures 6A-6E respectively show the ELISA detection of the binding reactions of SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 humanized antibodies to monkey MUC17 ECD3577-His protein.
  • 7A-7E are FACS detection of the binding reactions of SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 humanized antibodies to endogenous cells of NUGC4, respectively.
  • 8A-8E are FACS detection of the binding of SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 humanized antibodies to monkey MUC17 overexpressing cells FlpinCHO-Cyno(3597-3964)-D2, respectively.
  • compositions including A and B should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition”.
  • MUC17 refers to a member of the mucin family, which includes more than 20 members. Mucins are large, highly glycosylated membrane-bound proteins that are expressed almost exclusively in the gut. Their general function is to protect epithelial cells from the environment, as well as to regulate cell proliferation and survival. MUC17 is highly expressed in pancreatic adenocarcinoma tissues. MUC17 is expressed in pancreatic, appendix and some colon cancers. Its expression was not detectable in normal pancreas, pancreatitis, or cell lines derived from other cancers.
  • an antigen-binding molecule eg, an antibody
  • an antigen-binding molecule specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity.
  • Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity.
  • KD equilibrium dissociation constant
  • high affinity usually refers to having 1 ⁇ 10 -7 M or lower, about 1 ⁇ 10 -8 M or lower, about 1 ⁇ 10 -9 M or lower, about 1 ⁇ 10 -10 M or Lower, KD of 1 ⁇ 10 -11 M or lower or 1 ⁇ 10 -12 M or lower.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the on-rate.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg, Biacore) or equilibrium dialysis. For example, refer to the methods for obtaining the KD value shown in Example 3 or 6 herein.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes antibodies that do not comprise light chains, e.g., those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Vicugna heavy-chain antibodies (HCAbs) produced by camelids such as pacos and new immunoglobulin antigen receptors (IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Vicugna heavy-chain antibodies
  • heavy chain antibody refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • the term “nanobody” refers to the natural heavy chain antibody that lacks the light chain in camels, and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • single domain antibody single domain antibody, sdAb
  • CH1 light chain and heavy chain constant region 1
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • multispecific herein refers to having at least two antigen binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. combined.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody and single domain antibody.
  • Chimeric antibody herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity to target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the CDR of the variable region of the heavy chain can be abbreviated as HCDR
  • the CDR of the variable region of the light chain can be abbreviated as LCDR.
  • framework region or “FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • CDRs For further descriptions of CDRs, refer to Kabat et al., J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); People such as Chothia, J.Mol.Biol.196:901-917 (1987); People such as Al-Lazikani B., J.Mol.Biol., 273:927-948 (1997); People such as MacCallum, J.Mol .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • Fc refers to the carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole antibody, which typically includes the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the respective programs. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present application in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g.
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology, such as the progression of cancer, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject refers to an organism receiving treatment for a particular disease or condition as described herein.
  • subjects and patients include mammals, such as humans, primate (e.g., monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • the present application screened and obtained various nanobodies specifically binding to MUC17, and obtained various humanized forms of nanobodies by using these nanobodies. These nanobodies will be promising in the treatment of MUC17-expressing tumors.
  • the application provides a Nanobody specifically binding to MUC17 or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are respectively selected from SEQ ID NO: CDR1, CDR2 and CDR3 of the VHH shown in any one of ID NO.8-12, 62-66, 67-98.
  • the CDR1, CDR2 and CDR3 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, for example, the CDR1 comprises SEQ ID NO.13, 16, 19, 22, 25, 28, 31 , 34, 37, 40, 43, 46, 49, 52 or the amino acid sequence shown in 55;
  • the CDR2 comprises SEQ ID NO.14, 17, 20, 23, 26, 29, 32, 35, 38, 41, The amino acid sequence shown in 44, 47, 50, 53 or 56;
  • the CDR3 comprises SEQ ID NO.15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54 Or the amino acid sequence shown in 57.
  • CDR1 ⁇ 3 of VHH shown in SEQ ID NO.9, 63, 72 ⁇ 77 is according to IMGT, Kabat or Chothia numbering system, and has such as SEQ ID NO:16 ⁇ 18, SEQ ID NO:31 ⁇ 33 or SEQ ID NO : the amino acid sequence shown in 46-48;
  • CDR1 ⁇ 3 of VHH shown in SEQ ID NO.10, 64, 78 ⁇ 84 is according to IMGT, Kabat or Chothia numbering system, and has such as SEQ ID NO:19 ⁇ 21, SEQ ID NO:34 ⁇ 36 or SEQ ID NO : the amino acid sequence shown in 49-51;
  • CDR1 ⁇ 3 of VHH shown in SEQ ID NO.11, 65, 85 ⁇ 91 is according to IMGT, Kabat or Chothia numbering system, and has such as SEQ ID NO:22 ⁇ 24, SEQ ID NO:37 ⁇ 39 or SEQ ID NO : the amino acid sequence shown in 52-54;
  • CDR1 ⁇ 3 of VHH shown in SEQ ID NO.12, 66, 92 ⁇ 98 is according to IMGT, Kabat or Chothia numbering system, and has such as SEQ ID NO:25 ⁇ 27, SEQ ID NO:40 ⁇ 42 or SEQ ID NO : the amino acid sequence shown in 55-57.
  • said Nanobody or antigen-binding fragment thereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, compared to said CDR1, CDR2 and CDR3. %, 96%, 97%, 98%, 99% or 100% identity or CDR1, CDR2 and CDR3 sequences with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions, preferably, the Substitutions were made with conservative amino acid substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises a VHH shown in any one of SEQ ID NO.8-12, 62-66, 67-98, or with SEQ ID NO.
  • the VHH shown in any one of ⁇ 66, 67 ⁇ 98 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 VHH sequences of 1, 5, 4, 3, 2 or 1 mutations; the mutations can be selected from insertions, deletions and/or substitutions, and the substitutions are preferably conservative amino acid substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises a framework region sequence having at least a mutation selected from the group consisting of, numbering in natural order, compared to the framework region of the VHH set forth in SEQ ID NO: 62, H34G, V36F, G43E, L44R, W46G, S48A, G57I, R70K, Y93S, W114R or M119Q; preferably, at least H34G, V36F, G43E, L44R, W46G, S48A, G57I, R70K, Y93S and W114R mutations; more preferably Preferably, at least H34G, V36F, G43E, L44R, W46G, S48A and W114R mutations; more preferably, at least H34G, V36F, G43E, L44R, W46G, S48A, Y93S and W114R mutations; more preferably, at least H34G, V36F, G43E, L44R, W
  • Said Nanobody or antigen-binding fragment thereof comprises a framework region sequence having at least a mutation selected from the group consisting of at least a mutated framework region sequence compared to the framework region of the VHH shown in SEQ ID NO: 63: numbered in natural order, E1D, V5Q, E6A, G26D, F27N, V37F, G44E, L45R, W47G, R71Q, V92M, R97A or M118Q; preferably, at least V37F, G44E, L45R, W47G and R97A mutations; more preferably, at least V37F, G44E, L45R, W47G, R71Q and R97 mutations; more preferably at least F27N, V37F, G44E, L45R, W47G, R71Q and R97A mutations; more preferably at least E1D, G26D, F27N, V37F, G44E, L45R, W47G, R71Q and R97A mutations
  • Said Nanobody or antigen-binding fragment thereof comprises a framework region sequence having at least a mutation selected from the group consisting of at least a mutated framework region sequence compared to the framework region of the VHH shown in SEQ ID NO: 64: numbered in natural order, E1D, V5Q, E6A, T28P, F29S, V37F, G44E, L45R, W47G, K75L, N76S, Y79W, V92M, R97A or M118Q; preferably, at least V37F, G44E, L45R, W47G and R97A mutations; more preferably, at least T28P, F29S, V37F, G44E, L45R, W47G, and R97A mutations; more preferably, at least E1D, T28P, F29S, V37F, G44E, L45R, W47G, and R97A mutations; more preferably, at least E1D, T28P, F29S, V37F,
  • Said Nanobody or antigen-binding fragment thereof comprises a framework region sequence having at least a mutation selected from the group consisting of at least a mutated framework region sequence compared to the framework region of the VHH shown in SEQ ID NO: 65: numbered in natural order, E1D, V5Q, E6A, H35A, V37F, G44E, L45R, W47V, F68L, R72H, S75A, V93M or M119Q; preferably, at least H35A, V37F, G44E, L45R and W47V mutations; more preferably, at least H35A, V37F, G44E, L45R, W47V and R72H mutations; more preferably, at least E1D, H35A, V37F, G44E, L45R, W47V and R72H mutations; more preferably, at least E1D, H35A, V37F, G44E, L45R, W47V and R72H mutations; more preferably, at
  • the Nanobody or antigen-binding fragment comprises a framework region sequence with at least a mutation selected from the group consisting of, compared to the framework region of the VHH shown in SEQ ID NO: 66: numbered in natural order, V5Q, E6A, F27D, V37F , A40R, G44E, L45R, W47A, R71Q, S74T, V92M, K97A, or M118Q; preferably, at least V37F, G44E, L45R, W47A, and K97A mutations; more preferably, at least V37F, G44E, L45R, W47A, R71Q and K97A mutations; more preferably, at least F27D, V37F, G44E, L45R, W47A, R71Q and K97A mutations; more preferably, at least F27D, V37F, G44E, L45R, W47A, R71Q and K97A mutations; more preferably, at least F27D, V37
  • the Nanobody or antigen-binding fragment thereof specifically binds to human MUC17 protein and/or monkey MUC17 protein; preferably, its KD for binding to human MUC17 protein and/or monkey MUC17 protein is better than 1.00E -7M.
  • the Nanobody or antigen-binding fragment thereof is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.
  • the Nanobody or antigen-binding fragment thereof comprises or does not comprise an antibody heavy chain constant region; optionally, the antibody heavy chain constant region may be selected from human, alpaca, mouse, rat Mouse, rabbit or sheep; alternatively, the heavy chain constant region of the antibody can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the The heavy chain constant region may be selected from an Fc region, a CH3 region or a complete heavy chain constant region, preferably, the heavy chain constant region is a human Fc region; preferably, the Nanobody or an antigen-binding fragment thereof is a heavy chain antibody.
  • the antibody heavy chain constant region may be selected from human, alpaca, mouse, rat Mouse, rabbit or sheep; alternatively, the heavy chain constant region of the antibody can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be
  • the Nanobody or antigen-binding fragment thereof is further coupled to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of drugs, toxins, radioisotopes, chemotherapeutics or immunomodulators, so
  • the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the present application provides a multispecific molecule comprising any one of the above-mentioned Nanobodies or antigen-binding fragments thereof; preferably, the multispecific molecule further comprises specific binding to an antigen other than MUC17 or binding A Nanobody or an antigen-binding fragment thereof of a MUC17 epitope that is different from any of the Nanobodies or antigen-binding fragments thereof described above.
  • the antigen other than MUC17 is an antigen on the surface of T cells, B cells, natural killer cells, dendritic cells, macrophages, monocytes or neutrophils; preferably, the Antigens other than MUC17 are selected from: CD96, PD-1, PD-L1, PD-L2, OX40, OX40L, LAG-3, TIM3, VISTA, CD3, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD27, CD28, CD28H, CD16 , CD16A, CD32B, VEGF, NKG2D, NKp30, NKp46, NKp44, CD19, CD20, CD40, CD47, 4-1BB, ICOS, OX40, EGFR, EGFRvIII, TNF-alpha, CD33, HER2, HER3, HAS, CD5, CD27 , EphA2, EpCAM, MUC1, MUC16, CEA, Claudin18.2, folate receptor, Claudin
  • the multispecific molecule may be bispecific, trispecific or tetraspecific, more preferably, the multispecific molecule may be bivalent, tetravalent or hexavalent.
  • the multispecific molecule is a tandem scFv, diabody (Db), single chain diabody (scDb), dual affinity retargeting (DART) antibody, F(ab')2, dual Variable Domain (DVD) Antibodies, KiH Antibodies, Docking and Locking (DNL) Antibodies, Chemically Cross-Linked Antibodies, Heteropolymeric Nanobodies or Heteroconjugate Antibodies.
  • the application provides a chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain Comprising any one of the above Nanobodies or antigen-binding fragments thereof.
  • CAR chimeric antigen receptor
  • the application provides an immune effector cell expressing the chimeric antigen receptor, or comprising a nucleic acid fragment encoding the chimeric antigen receptor; preferably, the immune effector cell is selected from T cell, NK cell (natural killer cell), NKT cell (natural killer T cell), DNT cell (double negative T cell), monocyte, macrophage, dendritic cell or mast cell, said T cell is preferably From cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autologous immune effector cells or allogeneic immune effector cells.
  • the present application provides an isolated nucleic acid fragment encoding any one of the above Nanobodies or antigen-binding fragments thereof, or said multispecific molecule, or said chimeric antigen receptor.
  • the nucleic acid may be a codon-optimized nucleic acid suitable for expression in a host cell.
  • a codon-optimized nucleic acid suitable for expression in a host cell.
  • Methods for codon optimization depending on the host cell used are well known to those skilled in the art.
  • the present application provides a vector comprising the nucleic acid fragment.
  • the vector is an expression vector.
  • prokaryotic cloning vectors include plasmids from E. coli such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages.
  • An example of a vector that can be used in yeast is the 2 ⁇ plasmid.
  • Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences as well as those derived from functional mammalian vectors (such as those described above) and functional plasmids and Combinatorial shuttle vectors for phage DNA.
  • Expression vectors useful in this application contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of bacteriophage lambda, the control region of the fd coat protein, the glycolytic promoter of yeast, e.g.
  • the application provides a host cell comprising the vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells Animal cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells Animal cells (CHO cell line or 293T cell line).
  • the present application provides a method for preparing any one of the above-mentioned Nanobodies or antigen-binding fragments thereof or multispecific molecules, which comprises culturing said host cells, and isolating the Nanobodies or nanobodies expressed by said cells. Antigen-binding fragments, or isolated multispecific molecules expressed by said cells.
  • the present application provides a method for preparing the immune effector cells, which includes introducing the nucleic acid fragment encoding the CAR into the immune effector cells, and optionally, also including promoting the immune effector cells to express The CAR.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the above Nanobodies or antigen-binding fragments thereof, multispecific antibodies, immune effector cells, nucleic acid fragments, vectors, host cells, or any of the above
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the pharmaceutically acceptable carrier is a carrier that does not weaken the viability and function of immune cells, and does not affect the specific binding of the antibody or its antigen-binding fragment to the antigen, including but not limited to cell culture medium, buffer, Physiological saline and balanced salt solution, etc.
  • buffers include isotonic phosphate, acetate, citrate, borate, carbonate, and the like.
  • the pharmaceutically acceptable carrier is phosphate buffered saline containing 1% serum.
  • Nanobodies or antigen-binding fragments thereof disclosed herein and pharmaceutical compositions thereof can be used to treat, ameliorate or prevent tumors in individuals.
  • Nanobodies or antigen-binding fragments thereof disclosed herein and pharmaceutical compositions thereof may be administered in any suitable manner.
  • Nanobodies or antigen-binding fragments thereof of the present application and pharmaceutical compositions thereof are administered by injection (eg, subcutaneously, intravenously, intratumorally, intraarterially, intramuscularly, intradermally, intraperitoneally or intrathecally).
  • the Nanobodies or antigen-binding fragments thereof of the present application and pharmaceutical compositions thereof are administered intravenously.
  • suitable pharmaceutically acceptable carriers for injection may include any isotonic carrier, such as physiological saline (water containing about 0.90% w/v NaCl, Water containing about 300 mOsm/L NaCl, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), water containing about 5% glucose, or Lactated Ringer's solution.
  • human serum albumin is substituted for the pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further include a second agent for treating, improving or preventing individual tumors.
  • various chemotherapy drugs such as alkylating agent chemotherapy drugs, including but not limited to cyclophosphamide, ifosfamide, melphalan, carmustine, lomustine, nimustine, formol Sting, etc.; anti-metabolite chemotherapy drugs, including but not limited to methotrexate, fluorouracil, tegafur, carmofur, deoxyfluridine, capecitabine, gemcitabine, raltitrexed, etc.; anticancer antibiotics , including but not limited to actinomycin D, mitomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, pirarubicin, etc.; plant chemotherapy drugs, including but not Limited to vincristine, vindesine, vinorelbine, irinotecan, topotecan, paclitaxel, docetaxel, etc.
  • the present application provides a method for preventing and/or treating tumors, comprising administering to a patient in need thereof an effective amount of any one of the Nanobodies or antigen-binding fragments thereof, multispecific molecules, immune effectors Cells, nucleic acid fragments, vectors, host cells, products prepared by any of the above methods; or pharmaceutical compositions; wherein the tumor can be a tumor expressing MUC17 on the cell surface, such as a tumor involving the digestive system, such as gastric cancer, pancreatic cancer and/or gastroesophageal junction cancer.
  • the present application also provides any of the above Nanobodies or antigen-binding fragments thereof, multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, and products prepared by any of the above methods; or A pharmaceutical composition for preventing and/or treating tumors; wherein the tumors may be tumors expressing MUC17 on the cell surface, such as tumors involving the digestive system, such as gastric cancer, pancreatic cancer and/or gastroesophageal junction cancer.
  • the present application provides a kit comprising any of the above-mentioned Nanobodies or antigen-binding fragments thereof, multispecific antibodies, immune effector cells, nucleic acid fragments, vectors, host cells, any of the above-mentioned methods Preparation of the obtained product, or pharmaceutical composition.
  • the present application provides a method for detecting the expression of MUC17, wherein, under the condition that a complex can be formed between any of the above-mentioned Nanobodies or their antigen-binding fragments and MUC17, the sample to be detected and any of the above-mentioned A Nanobody or antigen-binding fragment thereof is contacted.
  • the present application provides a method for inhibiting the proliferation or migration of MUC17-expressing cells in vitro, wherein, under the condition that any one of the Nanobodies or antigen-binding fragments thereof and MUC17 can form a complex, the Said cell is contacted with any one of the Nanobodies or antigen-binding fragments thereof described above.
  • the amino acid sequence (UniProt: Q685J3) containing the truncation of the extracellular domain of the human MUC17 protein was cloned into the pTT5 vector with a His tag (Youbao Biology, VT2202), and the plasmid was prepared according to the method of the plasmid extraction kit. 293F cells (Gibco, A14527) were transiently expressed to obtain the antigens and detection proteins used in the Examples section.
  • the preparation method of the cynomolgus monkey MUC17 truncated extracellular region protein is the same as the preparation method of the human recombinant protein.
  • the cynomolgus monkey MUC17 sequence comes from Uniprot number: A0A2K5WH09, and the specific sequence information of the recombinant protein is as follows:
  • human MUC17 ECD4131-his His-tagged human MUC17 protein extracellular domain fusion protein
  • Cyno MUC17 ECD3577-his His-tagged cynomolgus monkey MUC17 protein extracellular domain fusion protein
  • control antibodies used in the examples are all from published patent sequences.
  • the 2D11 and 4C11 antibody sequences are derived from the published patent WO2019133961A1. Unless otherwise specified, the 2D11 and 4C11 control antibodies are recombined using human IgG1+ ⁇ subtype Express. Nanobodies and their humanized antibodies used in the examples are all recombinantly expressed in the form of human Fc fusion.
  • the expression and purification process of the control antibody was as follows: the antibody sequence gene was synthesized and cloned into the expression vector pTT5, then transiently transfected into Expi293F cells (purchased from Gibco, A14527), cultured on a shaker at 37°C for 7 days, and the supernatant of the cells was collected for protein A
  • Expi293F cells purchased from Gibco, A14527
  • the resulting control antibodies were named 2D11-hIgG1 and 4C11-hIgG1.
  • the specific sequence information of the antibody is shown in Table 1.
  • step "1.1 Design and Expression of Recombinant Proteins” After constructing and expressing related recombinant proteins according to step "1.1 Design and Expression of Recombinant Proteins", purify them as follows: centrifuge the cell expression supernatant samples at high speed to remove impurities, equilibrate the nickel column with 20mM PBS+500mM NaCl solution, wash 2- 5 column volumes.
  • the culture supernatant was loaded onto a Ni affinity chromatography column (purchased from GE Healthcare), while an ultraviolet (UV) detector was used to monitor changes in the UV absorbance (A280nm), and the column was washed with an equilibrium solution until the A280 reading dropped to Baseline, followed by gradient elution with different concentrations of imidazole, collect each elution peak, and determine the component of the target protein according to the SDS-PAGE gel map.
  • the eluted product was collected, concentrated and further purified by gel chromatography Superdex200 (GE). The obtained protein was separated and used after the purity test by electrophoresis and HPLC was qualified.
  • the proteins purified by this protocol include human MUC17-His and monkey MUC17-His.
  • the obtained antibody sequences were respectively cloned into the eukaryotic expression vector pTT5 with human Fc tag, and Expi293F cells were transiently transfected with PEI (Polysciences, 24765-1). After 7 days of culture, the cell culture supernatant expressing the antibody was collected by high-speed centrifugation. Wash the Protein A (Borgeron, AA0273) protein chromatography column with 3-5 column volumes of 0.1M NaOH, and then with 3-5 column volumes of pure water. Use 3-5 times column volume of 1 ⁇ PBS (pH7.4) buffer system as the equilibration buffer to equilibrate the chromatography column.
  • 1 ⁇ PBS pH7.4
  • the cell supernatant is combined with the sample at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer.
  • the chromatographic column is washed with 3-5 times the column volume of 1 ⁇ PBS (pH7.4) until the UV absorption drops to baseline.
  • the eluted product can be replaced by a method well known to those skilled in the art, such as using an ultrafiltration tube for ultrafiltration concentration and solution replacement to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required A buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove aggregate components in the eluted product to improve sample purity.
  • a high-resolution molecular exclusion column such as Superdex 200 to remove aggregate components in the eluted product to improve sample purity.
  • the antibody with human Fc tag eluted from the chromatography column was collected to obtain the corresponding control antibody.
  • Enzyme-linked immunosorbent assay was used to detect the titer and specificity of antibodies against human and monkey MUC17 proteins in the serum.
  • the serum titer results of the fourth immunization are shown in Table 2, and the data in the table are OD450nm values.
  • SfiI restriction enzyme
  • This patent adopts the method of cross-panning human and monkey MUC17 proteins.
  • For the first round of biopanning prepare tubes A, B, and C. Add 100 ⁇ L of streptavidin-coupled Dynabeads (purchased from Invitrogen) and the above-mentioned phage antibody library to tube A, and add 100 ⁇ L to tube B. Streptavidin-conjugated Dynabeads. Then add blocking solution, namely PBS phosphate buffer solution containing 20% (w/v) skimmed milk powder, to the three tubes respectively, and block at room temperature for 2 hours.
  • blocking solution namely PBS phosphate buffer solution containing 20% (w/v) skimmed milk powder
  • PBST the blocking solution containing 0.01% (v/v) Tween-20
  • PBS buffer 500 ⁇ l of 10 ⁇ g/mL trypsin was added to each tube and incubated at 37°C for 15 minutes to elute the phage bound to the biotinylated human MUC17-His protein.
  • 250 ⁇ l of trypsin solution was added to 4 mL of Escherichia coli TG1 (purchased from LUCIGEN) in logarithmic growth phase, and incubated at 37° C. for 30 minutes to obtain TG1 culture solution.
  • the culture solution of TG1 was serially diluted, spread on the plate, and cultured overnight at 37°C. Calculate the number of clones that bind to the biotinylated human MUC17-His protein and the control tube, and select 48 clones for sequencing.
  • the clones on the plate were washed and collected with 2YT medium (purchased from Sangong, the preparation method of 2YT medium is: add 31g of 2YT medium powder into 1L of water, adjust the pH to 7.0 with NaOH, and autoclave). And inoculated into fresh medium, 37 °C cultivated to the logarithmic phase.
  • helper phage M13KO7 (purchased from NEB, product number N0315S), the ratio of helper phage to Escherichia coli TG1 is 20:1, mix well, and stand at 37°C for 30 minutes. Then shake culture at 37°C for 30 minutes, collect cells after centrifugation at 4000rpm for 10 minutes, add fresh 2YT medium containing ampicillin and kana resistance, and shake culture at 30°C overnight. Centrifuge at 5000 rpm for 20 minutes, collect the supernatant, add 1/4 volume of the supernatant to 2.5M NaCl solution containing 20% PEG6000, and place on ice overnight.
  • the steps of the second round of panning were the same as the first round, and the second round enriched the VHH antibodies specifically binding to the biotinylated monkey MUC17-His protein.
  • the positive phages were continuously enriched during the panning process in order to screen for nanobodies with good specificity and high affinity.
  • Single clones were selected from the second and third rounds of plates and cultured in 96-well plates, and 200 ⁇ L of 2YT medium containing antibiotics and 1% glucose was added to each well, and cultured overnight at 37° C. and 250 rpm with shaking. Take 10 ⁇ L of the overnight culture and add it to 100 ⁇ l of 2YT medium containing antibiotics and 0.5% glucose, cultivate until the OD600 is 0.4-0.6, add helper phage at an infection ratio of 20:1, and let stand at 37°C for 30 minutes. Then shake culture at 37°C for 30 minutes, then add 400 ⁇ l of antibiotic-containing 2YT medium, and culture at 30°C overnight. The next day, centrifuge at 5000 rpm at 4°C for 20 minutes, and the supernatant obtained is used for monoclonal ELISA identification.
  • Human and monkey MUC17 proteins were diluted with carbonate buffer solution with a pH value of 9.6 to a final concentration of 2 ⁇ g/mL, added to enzyme-labeled wells at 50 ⁇ L/well, and coated overnight at 4°C. Then 50 ⁇ L of phage culture supernatant and 1:4000 diluted horseradish peroxidase-labeled anti-M13 antibody (purchased from Yiqiao Shenzhou, 11973-MM05T-H) were added to each well, and TMB chromogenic solution (purchased The color was developed from KPL, 52-00-02), and the optical density was measured at 450nm. The positive clones that combined human and monkey MUC17 proteins were selected for FACS detection.
  • the nucleotide sequence encoding the monkey MUC17 fragment was cloned into the pcDNA5 vector (purchased from Universal), the plasmid was prepared and an overexpression cell line was constructed, and the monoclonal cell line with higher fluorescence intensity was selected for subsequent detection.
  • the constructed overexpression cell line was named: FlpinCHO-Cyno(3597-3964)-D2.
  • Endogenous tumor cell line NUGC4 (Nanjing Kebai, catalog number: CBP60493) and FlpinCHO-Cyno(3597-3964)-D2 overexpressed cell line were respectively expanded and cultured in T-175 cell culture flasks to 90% confluence.
  • the medium was aspirated, washed once with PBS buffer, and then treated with Trypsin-EDTA (purchased from Gibco, catalog number 25200072) to collect the cells. After cell counting, the cells were washed twice with PBS phosphate buffer, diluted to 2 ⁇ 10 6 cells per milliliter, and added to a 96-well FACS reaction plate at 50 ⁇ L per well.
  • 1% (w/w) fetal calf serum was added to PBS phosphate buffer as FACS buffer, and centrifuged at 1000 rpm at 4° C. to wash twice. Add 50 ⁇ L of phage supernatant to each well and incubate on ice for 1 hour. Centrifuge and wash with FACS buffer for 3 times, add 50 ⁇ L of 1:1000 diluted anti-M13 antibody (Sino Biological, product number 11973-MM05T) to each well, incubate on ice for 1 hour, wash with FACS buffer for 3 times, add Fluorescent (Alexa 647)-labeled secondary antibody (Jackson Immuno, Cat. No. 115-605-003), incubated on ice for 1 hour. Wash 3 times by centrifugation with FACS buffer. The cells were suspended with 100 ⁇ L of FACS buffer, and the results were detected and analyzed by FACS.
  • the obtained nanobody sequences were respectively cloned into the eukaryotic expression vector pTT5 with Fc tag, and Expi293F cells (purchased from Gibco, A14527) were transiently transfected with PEI, cultured for 7 days, and the cell culture supernatant expressing the antibody was collected by high-speed centrifugation.
  • the antibodies were purified according to the purification method described in Example 1.3.2 to obtain the corresponding recombinant nanobodies, which were named Lab315-huFc, Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc respectively. Use the SEC-HPLC method to test the purity of the purified antibody, and distribute it for use after passing the test.
  • Enzyme-linked immunosorbent assay to detect the binding of recombinant antibody to human MUC17-his protein
  • Human MUC17-his protein was diluted with PBS to a final concentration of 2 ⁇ g/mL, then added to a 96-well ELISA plate at 50 ⁇ l per well, and incubated overnight at 4°C. The next day, the plate was washed twice with PBST, and a blocking solution [PBS+2% (w/w) BSA] was added to block at room temperature for 2 hours. Pour off the blocking solution, add 100nM as the initial concentration, 3-fold serially diluted recombinant antibody, and 50 ⁇ l of positive and negative control antibodies per well. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST.
  • HRP horseradish peroxidase-labeled secondary antibody (purchased from Merck, product number: AP113P), incubate at 37° C. for 1 hour, and wash the plate 5 times with PBST. 50 ⁇ l of TMB substrate was added to each well, and after incubation at room temperature for 10 minutes, 50 ⁇ l of stop solution (1.0 M HCl) was added to each well.
  • Enzyme-linked immunosorbent assay to detect the binding of recombinant antibody to monkey MUC17-his
  • the monkey MUC17-his protein was detected by ELISA and analyzed according to the method in Example 3.2.
  • the analysis results are shown in Figure 2A-2B, the recombinant antibodies Lab315-huFc, Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc have good binding activity to monkey MUC17-his protein.
  • the endogenous tumor cell NUGC4 was expanded to the logarithmic growth phase in a T-175 culture flask, the medium was aspirated, washed twice with PBS buffer, the cells were digested with trypsin, then the digestion was terminated with complete medium, and pipetting cells to a single cell suspension.
  • FACS buffer PBS+2% fetal bovine serum
  • the recombinant antibodies Lab315-huFc, Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc could specifically bind to NUGC-4 cells.
  • the FlpinCHO-Cyno(3597-3964)-D2 recombinant cells were subjected to FACS detection and data analysis according to the method in Example 3.4. The results are shown in Figures 4A-4B, the recombinant antibodies Lab315-huFc, Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc can effectively bind to FlpinCHO-Cyno(3597-3964)-D2 cells.
  • the BIAcore 8K instrument was used to detect the binding strength of the antibody to the antigen using the Protein A capture method.
  • protein A was immobilized on a CM4 chip (purchased from GE, BR-1005-34) by amino coupling method, and was mixed with HBS-EP+pH7. 4 is the mobile phase.
  • NHS and EDC activate the chip for about 600 seconds, dilute Protein A to 50 ⁇ g/mL with 10 mM sodium acetate pH4.5, inject for 600 seconds, and finally block the remaining activation sites with ethanolamine. Then, the affinity of the antibody to the antigen is determined by a multi-cycle kinetic method.
  • the protein A chip is used to capture the antibody to be tested, and then a single concentration of the antigen protein is injected to record the binding and dissociation process of the antibody and the antigen protein.
  • the chip regeneration was completed with Glycine pH1.5, where the mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate was 30 ⁇ L/min, the regeneration time was 30s, and the detection temperature was 25°C; , according to the 1:1binding model, analyze the data, and fit the antibody-antigen binding kinetic parameters, including the binding rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.
  • the binding rate (Ka), dissociation rate (Kd) and binding affinity (KD) of recombinant antibodies Lab315-huFc, Lab316-huFc, Lab320-huFc, Lab331-huFc and Lab332-huFc to human MUC17 protein are shown in Table 5.
  • the heavy chain variable region germline genes with high homology to Nanobodies were selected as templates, and the Nanobody
  • the CDRs sequences of the antibody based on the IMGT or KABAT nomenclature are grafted into the corresponding human templates to form variable region sequences in the order "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4".
  • the CDR sequence of the Lab315 antibody is defined based on the IMGT rules. Its humanized templates are IGHV3-9*01 and IGHJ3*01.
  • the Lab315 antibody was renamed as SCR-7724 during the humanization process, and after the CDR transplantation, it was named as SCR-7724- 001-H.
  • the CDR sequence of the Lab316 antibody is defined based on the Kabat rules. Its humanized templates are IGHV3-7*01 and IGHJ3*01.
  • the Lab316 antibody was renamed as SCR-7782 during the humanization process, and after the CDR transplantation, it was named as SCR-7782- 001-H.
  • the CDR sequence of the Lab320 antibody is defined based on the Kabat rules, and its humanized templates are IGHV3-7*01 and IGHJ3*01.
  • the Lab320 antibody was renamed as SCR-7783 during the humanization process, and after CDR transplantation, it was named as SCR-7783- 001-H.
  • the CDR sequence of the Lab331 antibody is defined based on the IMGT rules. Its humanized templates are IGHV3-30*02 and IGHJ3*01.
  • the Lab331 antibody was renamed SCR-7784 during the humanization process, and the CDR was named SCR-7784- 001-H.
  • the CDR sequence of the Lab332 antibody is defined based on the Kabat rules, and its humanized templates are IGHV3-30*02 and IGHJ3*01.
  • the Lab332 antibody was renamed as SCR-7785 during the humanization process, and after the CDR transplantation, it was named as SCR-7785- 001-H.
  • the CDRs of antibodies SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 were respectively grafted into their humanized templates to obtain humanized antibodies.
  • the amino acid sequence of the humanized template and the sequence of the humanized antibody after CDR graft are shown in Table 6.
  • the key amino acids in the FR region sequence of the SCR-7724 humanized antibody were back-mutated to the corresponding amino acids of the camel antibody to ensure the original affinity.
  • the details of the mutation points after the back-mutation (the back-mutation points are numbered in natural order) and The specific amino acid sequence is shown in Table 7-8.
  • Graft (IGHV3-9*01) represents the implantation of Nanobody CDR into the human germline FR region sequence; H34G represents the mutation of the 34th H of Graft (IGHV3-9*01) to G, and so on.
  • the key amino acids in the FR region sequence of the SCR-7782 humanized antibody were back-mutated to the amino acids corresponding to the camel antibody to ensure the original affinity.
  • the details of the mutation points after the back-mutation (the back-mutation points are numbered in natural order) and The specific amino acid sequence is shown in Table 9-10.
  • Graft (IGHV3-7*01) means that Nanobody CDR is implanted into the human germline FR region sequence; V37F means that the 37th V of Graft (IGHV3-7*01) is mutated to F, and so on.
  • the key amino acids in the FR region sequence of the SCR-7783 humanized antibody were back-mutated to the corresponding amino acids of the camel antibody to ensure the original affinity.
  • the details of the mutation points after the back-mutation (the back-mutation points are numbered in natural order) and The specific amino acid sequence is shown in Table 11-12.
  • Graft (IGHV3-7*01) means that Nanobody CDR is implanted into the human germline FR region sequence; V37F means that the 37th V of Graft (IGHV3-7*01) is mutated to F, and so on.
  • the key amino acids in the FR region sequence of the SCR-7784 humanized antibody were back-mutated to the corresponding amino acids of the camel antibody to ensure the original affinity.
  • the details of the mutation points after the back-mutation (the back-mutation points are numbered in natural order) and The specific amino acid sequence is shown in Table 13-14.
  • Graft (IGHV3-30*02) means that Nanobody CDR is implanted into the human germline FR region sequence; H35A means that the 35th H of Graft (IGHV3-30*02) is changed to A, and so on.
  • the CDR sequences of the SCR-7785 antibody were defined based on Kabat rules.
  • the key amino acids in the FR region sequence of the SCR-7785 humanized antibody were back-mutated to the corresponding amino acids of the camel antibody to ensure the original affinity.
  • the details of the mutation points after the back-mutation (the back-mutation points are numbered in natural order) and The specific amino acid sequence is shown in Table 15-16.
  • Graft (IGHV3-30*02) means that the camel antibody CDR was implanted into the human germline FR region sequence; V37F means that the 37th V of Graft (IGHV3-30*02) was mutated to F, and so on.
  • the humanized antibody variable region sequence gene was synthesized, it was cloned into the pTT5 vector with human hinge region and Fc constant region sequence to form a VHH-huFc (C220S) expression sequence, and a plasmid was prepared.
  • the antibody plasmid was transiently transfected into Expi293F cells by PEI, and the supernatant was collected after 7 days of culture, and the antibody was purified by protein A according to the method in Example 1.3.2.
  • Example 3.2 Refer to the method steps in Example 3.2 to detect the binding activity of the humanized antibody to the human MUC17-his protein.
  • the experimental results are shown in Figures 5A-5E.
  • the humanized antibodies SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 all maintained the binding ability of recombinant antibodies, and had good binding ability with human MUC17 protein. binding activity.
  • the monkey MUC17-his protein was diluted with PBS to a final concentration of 2 ⁇ g/mL, and ELISA detection and data analysis were performed according to the method in Example 3.3.
  • the results are shown in Figures 6A-6E, the humanized antibodies SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 have cross-binding activity with monkey MUC17 protein, maintaining the binding ability of the recombinant antibody.
  • the preparation of monkey MUC17 overexpressing cell FlpinCHO-Cyno(3597-3964)-D2 and the FACS detection method refer to Examples 3.4 and 3.5.
  • the experimental results are shown in Figures 8A-8E, the humanized antibodies SCR-7724, SCR-7782, SCR-7783, SCR-7784 and SCR-7785 in this example can all be combined with FlpinCHO-Cyno(3597-3964)- D2 recombinant cells have better cross-binding activity and maintain the equivalent binding ability of recombinant antibodies.

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Abstract

一种能够特异性地结合MUC17的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够以高亲和力与MUC17特异性结合，可作为药物治疗胃肠道恶性肿瘤。

Description

抗MUC17纳米抗体及其应用
相关申请的交叉引用
本申请要求于2021年10月21日提交的申请号为202111224424.2的中国专利申请的优先权权益，其通过引用整体并入本文。
技术领域
本申请涉及抗体领域，具体而言，涉及抗MUC17纳米抗体。
背景技术
驼科动物和软骨鱼体内存在一种缺失轻链的特殊抗体，通常把源于这种抗体的可变区片段称为纳米抗体(Nanobody)，纳米抗体一般CDR3较长，可形成稳定的大凸环结构，能够结合一些隐蔽的抗原表位，特别适用于比较难得到抗体的靶点，如GPCR、离子通道和酶活中心等。与传统抗体相比，VHH纳米抗体分子量小，易于体外表达，可溶性好，免疫原性弱，能够穿过机体内的一些保护性屏障进入发病部位发挥作用，如血脑屏障等。
粘蛋白是一个高分子量糖蛋白家族，可分为分泌蛋白和跨膜蛋白，其富含丝氨酸、苏氨酸的蛋白骨架与多种O-型寡聚糖侧链结合，典型的寡聚糖链占成熟粘蛋白的70％左右。膜结合型粘蛋白即细胞表面粘蛋白，依靠跨膜区与细胞膜结合，成员包含MUC1、MUC3A、MUC3B、MUC4、MUC12、MUC13、MUC15、MUC16和MUC17。MUC17(Mucin-17)是一种单次跨膜粘蛋白，与MUC3的同源性较高，于2002年首次发现。MUC17蛋白全长4493个氨基酸，胞外段包含59个富含丝氨酸、苏氨酸和脯氨酸的串联重复序列、2个EGF和1个SEA结构域，胞内段含有80个氨基酸胞质尾和磷酸化位点，胞内段的磷酸化修饰可能与细胞增殖和凋亡的信号传导有关。不同类型的膜结合型粘蛋白其表达的部位也不同，例如在正常人体，MUC1主要表达于胃，MUC17则主要表达于肠道。在肿瘤组织中，MUC17同时在胃癌和胰腺癌高表达，属于肿瘤相关抗原。靶向MUC17的药物开发对胃癌和胰腺癌的临床治疗有重要意义。
目前，针对MUC17靶点只有Amgen双特异性抗体AMG199获批临床，用于治疗MUC17阳性的胃癌或胃食管交界部癌，其通过同时与T细胞上的CD3和肿瘤细胞上的MUC17结合，产生针对表达MUC17肿瘤细胞的细胞毒作用。
发明内容
本申请筛选获得MUC17特异的纳米抗体，拟用于治疗不同形式的恶性肿瘤。本申请提供抗MUC17抗体，编码其的核酸，抗体制备方法，含有所述抗体的药物组合物，以及药物组合物用于治疗肿瘤的相关用途。
在第一方面，本申请提供了一种特异性结合MUC17的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别选自SEQ ID NO.8～12、62～66、67～98中任一项所示的VHH的CDR1、CDR2和CDR3。
在第二方面，本申请提供了一种多特异性分子，其包含第一方面所述的纳米抗体或其抗原结合片段。
在第三方面，本申请提供了一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含第一方面所述的纳米抗体或其抗原结合片段。
在第四方面，本申请提供了一种免疫效应细胞，其表达第三方面所述的嵌合抗原受体，或包含编码第三方面所述的嵌合抗原受体的核酸片段。
在第五方面，本申请提供了一种分离的核酸片段，其编码第一方面所述的纳米抗体或其抗原结合片段，或第二方面所述的多特异性分子，或第三方面所述的嵌合抗原受体。
在第六方面，本申请提供了一种载体(vector)，其包含第五方面所述的核酸片段。
在第七方面，本申请提供了一种宿主细胞，其包含第六方面所述的载体。
在第八方面，本申请提供了一种制备第一方面所述的纳米抗体或其抗原结合片段或第二方面所述的多特异性分子的方法，其包括培养第七方面所述的细胞，以及分离所述细胞表达的纳米抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
在第九方面，本申请提供了一种制备第四方面所述的免疫效应细胞的方法，其包括将编码第三方面所述的CAR的核酸片段导入所述免疫效应细胞。
在第十方面，本申请提供了一种药物组合物，其包含第一方面所述的纳米抗体或其抗原结合片段，第二方面所述的多特异性抗体，第四方面所述的免疫效应细胞，第五方面的核酸片段，第六方面所述的载体，第七方面所述的宿主细胞，或第八方面所述的方法制备获得的产品。
在第十一方面，本申请提供了第一方面所述的纳米抗体或其抗原结合片段，第二方面所述的多特异性抗体，第四方面所述的免疫效应细胞，第五方面的核酸片段，第六方面所述的载体，第七方面所述的宿主细胞，第八方面所述的方法制备获得的产品，或第十方面所述的药物组合物在制备预防和/或治疗肿瘤的药物中的用途。
在第十二方面，本申请提供了一种预防和/或治疗肿瘤的方法，包括向有此需要的患者施用有效量的第一方面所述的纳米抗体或其抗原结合片段，第二方面所述的多特异性抗体，第四方面所述的免疫效应细胞，第五方面的核酸片段，第六方面所述的载体，第七方面所述的宿主细胞，第八方面所述的方法制备获得的产品，或第十方面所述的药物组合物。
在第十三方面，本申请提供了第一方面所述的纳米抗体或其抗原结合片段，第二方面所述的多特异性抗体，第四方面所述的免疫效应细胞，第五方面的核酸片段，第六方面所述的载体，第七方面所述的宿主细胞，第八方面所述的方法制备获得的产品，或第十方面所述的药物组合物用于预防和/或治疗肿瘤的用途。
在第十四方面，本申请提供了一种试剂盒，其包含第一方面所述的纳米抗体或其抗原结合片段，第二方面所述的多特异性抗体，第四方面所述的免疫效应细胞，第五方面的核酸片段，第六方面所述的载体，第七方面所述的宿主细胞，第八方面所述的方法制备获得的产品，或第十方面所述的药物组合物。
在第十五方面，本申请提供了一种检测MUC17表达的方法，其中在第一方面所述的纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使待检测样品与第一方面所述的纳米抗体或其抗原结合片段接触。
在第十六方面，本申请提供了一种体外抑制表达MUC17的细胞增殖或迁移的方法，其 中，在第一方面所述的纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使所述细胞与第一方面所述的纳米抗体或其抗原结合片段接触。
附图说明
图1A为ELISA检测Lab315-huFc重组抗体与人MUC17 ECD4131-his蛋白结合反应。
图1B为ELISA检测Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc重组抗体与人MUC17 ECD4131-his蛋白结合反应。
图2A为ELISA检测Lab315-huFc重组抗体与猴MUC17 ECD3577-His蛋白结合反应。
图2B为ELISA检测Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc重组抗体与猴MUC17 ECD3577-His蛋白结合反应。
图3A为FACS检测Lab315-huFc重组抗体与NUGC4内源肿瘤细胞结合反应。
图3B为FACS检测Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc重组抗体与NUGC4内源肿瘤细胞结合反应。
图4A为FACS检测Lab315-huFc重组抗体与猴MUC17过表达细胞FlpinCHO-Cyno(3597-3964)-D2的结合。
图4B为FACS检测Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc重组抗体与猴MUC17过表达细胞FlpinCHO-Cyno(3597-3964)-D2的结合。
图5A-5E分别为ELISA检测SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785人源化抗体与人MUC17 ECD4131-his蛋白结合反应。
图6A-6E分别为ELISA检测SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785人源化抗体与猴MUC17 ECD3577-His蛋白结合反应。
图7A-7E分别为FACS检测SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785人源化抗体与NUGC4内源细胞结合反应。
图8A-8E分别为FACS检测SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785人源化抗体与猴MUC17过表达细胞FlpinCHO-Cyno(3597-3964)-D2的结合。
发明的详细描述
术语定义和说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
本文术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全 部或任何其他组合”的含义。
本文术语“MUC17”是指粘蛋白家族的成员，粘蛋白家族包括超过20个成员。粘蛋白是大的、高度糖基化的膜结合蛋白，它们几乎只在肠道中表达。它们的一般功能是保护上皮细胞免受环境影响，以及调节细胞的增殖和存活。MUC17在胰腺腺癌组织中高度表达。MUC17在胰腺癌、阑尾癌和一些结肠癌中表达。在正常胰腺、胰腺炎或源自其他癌症的细胞系中检测不到其表达。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有1×10 -7M或更低、约1×10 -8M或更低、约1×10 -9M或更低、约1×10 -10M或更低、1×10 -11M或更低或1×10 -12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定，示例性地，可参见本文实施例3或6所示KD值获得方法。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
如本文所用，术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗 体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，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。
关于“重链抗体”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’) 2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全 人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本申请的核酸(例如编码SEQ ID NO:1的核酸)分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本申请的蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或 轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本申请的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2 101 823B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本申请所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵 长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
具体实施方案
本申请筛选获得了特异性结合MUC17的多种纳米抗体，并利用这些纳米抗体获得了多种人源化形式的纳米抗体。这些纳米抗体将在治疗表达MUC17的肿瘤方面具有应用前景。
具体而言，本申请提供了如下技术方案：
第一个方面，本申请提供了一种特异性结合MUC17的纳米抗体或其抗原结合片段，其中所述抗体或抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别选自SEQ ID NO.8～12、62～66、67～98中任一项所示的VHH的CDR1、CDR2和CDR3。
在一些实施方案中，所述CDR1、CDR2和CDR3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定，例如，所述CDR1包含SEQ ID NO.13、16、19、22、25、28、31、34、37、40、43、46、49、52或55所示的氨基酸序列；所述CDR2包含SEQ ID NO.14、17、20、23、26、29、32、35、38、41、44、47、50、53或56所示的氨基酸序列；所述CDR3包含SEQ ID NO.15、18、21、24、27、30、33、36、39、42、45、48、51、54或57所示的氨基酸序列。
在一些实施方案中，SEQ ID NO.8、62、67～71所示的VHH的CDR1、CDR2和CDR3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：13～15、SEQ ID NO：28～30或SEQ ID NO：43～45所示的氨基酸序列；
SEQ ID NO.9、63、72～77所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：16～18、SEQ ID NO：31～33或SEQ ID NO：46～48所示的氨基酸序列；
SEQ ID NO.10、64、78～84所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：19～21、SEQ ID NO：34～36或SEQ ID NO：49～51所示的氨基酸序列；
SEQ ID NO.11、65、85～91所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：22～24、SEQ ID NO：37～39或SEQ ID NO：52～54所示的氨基酸序列；
SEQ ID NO.12、66、92～98所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：25～27、SEQ ID NO：40～42或SEQ ID NO：55～57所示的氨基酸序列。
在一些实施方案中，所述纳米抗体或其抗原结合片段包含与所述CDR1、CDR2和CDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDR1、CDR2和CDR3序列，优选地，所述替换为保守氨基酸的替换。
在一些实施方案中，所述纳米抗体或其抗原结合片段包含SEQ ID NO.8～12、62～66、67～98任一项所示的VHH，或者与SEQ ID NO.8～12、62～66、67～98任一项所示的VHH具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性或至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的VHH序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些实施方案中，所述纳米抗体或其抗原结合片段包含与SEQ ID NO：62所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，H34G，V36F，G43E，L44R，W46G，S48A，G57I，R70K，Y93S，W114R或M119Q；优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，G57I，R70K，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，R70K，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，R70K，Y93S，W114R和M119Q突变；
所述纳米抗体或其抗原结合片段包含与SEQ ID NO：63所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，G26D，F27N，V37F，G44E，L45R，W47G，R71Q，V92M，R97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有V37F，G44E，L45R，W47G，R71Q和R97突变；更优选地，至少具有F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，G26D，F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，V5Q，E6A，G26D，F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，G26D，F27N，V37F，G44E，L45R，W47G，R71Q，V92M，R97A和M118Q突变；
所述纳米抗体或其抗原结合片段包含与SEQ ID NO：64所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S，Y79W，V92M，R97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有T28P，F29S，V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E， L45R，W47G，K75L，N76S，Y79W和R97A突变；更优选地，至少具有E1D，V5Q，E6A，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S，V92M，R97A和M118Q突变；
所述纳米抗体或其抗原结合片段包含与SEQ ID NO：65所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，H35A，V37F，G44E，L45R，W47V，F68L，R72H，S75A，V93M或M119Q；优选地，至少具有H35A，V37F，G44E，L45R和W47V突变；更优选地，至少具有H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，R72H和S75A突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，F68L，R72H和S75A突变；更优选地，至少具有E1D，V5Q，E6A，H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，R72H，V93M和M119Q突变；
或所述纳米抗体或抗原结合片段包含与SEQ ID NO：66所示VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，V5Q，E6A，F27D，V37F，A40R，G44E，L45R，W47A，R71Q，S74T，V92M，K97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47A和K97A突变；更优选地，至少具有V37F，G44E，L45R，W47A，R71Q和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q，S74T和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q，V92M，K97A和M118Q突变；更优选地，至少具有F27D，V37F，A40R，G44E，L45R，W47A，R71Q，V92M，K97A和M118Q突变；更优选地，至少具有V5Q，E6A，F27D，V37F，G44E，L45R，W47A，R71Q和K97A突变。
在一些实施方案中，所述纳米抗体或其抗原结合片段与人MUC17蛋白和/或猴MUC17蛋白特异性结合；优选地，其与人MUC17蛋白和/或猴MUC17蛋白结合的KD优于1.00E-7M。
在一些实施方案中，所述纳米抗体或其抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些实施方案中，所述纳米抗体或其抗原结合片段，其包含或不包含抗体重链恒定区；可选的，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述纳米抗体或其抗原结合片段为重链抗体。
在一些实施方案中，所述纳米抗体或其抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在另一方面，本申请提供了一种多特异性分子，其包含上述任一项纳米抗体或其抗原结合片段；优选地，所述多特异性分子进一步包含特异性结合MUC17以外的抗原或结合与上 述任一项纳米抗体或其抗原结合片段不同的MUC17表位的纳米抗体或其抗原结合片段。
在一些实施方案中，所述MUC17以外的抗原为T细胞、B细胞、自然杀伤细胞、树突状细胞、巨噬细胞、单核细胞或嗜中性细胞表面上的抗原；优选地，所述MUC17以外的抗原选自：CD96、PD-1、PD-L1、PD-L2、OX40、OX40L、LAG-3、TIM3、VISTA、CD3、CD3γ、CD3δ、CD3ε、CD3ζ、CD27、CD28、CD28H、CD16、CD16A、CD32B、VEGF、NKG2D、NKp30、NKp46、NKp44、CD19、CD20、CD40、CD47、4-1BB、ICOS、OX40、EGFR、EGFRvIII、TNF-alpha、CD33、HER2、HER3、HAS、CD5、CD27、EphA2、EpCAM、MUC1、MUC16、CEA、Claudin18.2、叶酸受体、Claudin6、WT1、NY-ESO-1、MAGE3、ASGPR1、TGFβ-trap、IL-2、IL-15、IL-21、IL-18或CDH16；
优选地，所述多特异性分子可为双特异性、三特异性或四特异性，更优选地，所述多特异性分子可为二价、四价或六价。
在一些实施方案中，所述多特异性分子为串联scFv、双功能抗体(Db)、单链双功能抗体(scDb)、双重亲和力再靶向(DART)抗体、F(ab')2、双重可变域(DVD)抗体、臼包杵(KiH)抗体、对接及锁定(DNL)抗体、化学交联抗体、杂多聚纳米抗体或异结合物抗体。
在另一方面，本申请提供了一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述任一种纳米抗体或其抗原结合片段。
在另一方面，本申请提供了一种免疫效应细胞，其表达所述的嵌合抗原受体，或包含编码所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
在另一方面，本申请提供了一种分离的核酸片段，其编码上述任一种纳米抗体或其抗原结合片段，或所述多特异性分子，或所述嵌合抗原受体。
在优选的实施方案中，所述核酸可以是适合在宿主细胞中表达的密码子优化的核酸。例如根据密码子的简并性，其仍然编码同样的蛋白质。根据所用宿主细胞进行密码子优化的方法是本领域技术人员公知的。
在另一方面，本申请提供了一种载体(vector)，其包含所述核酸片段。优选地，所述载体为表达载体。
可以使用任何合适的表达载体。例如，原核克隆载体包括来自大肠杆菌的质粒，如colEl、pCRl、pBR322、pMB9、pUC、pKSM和RP4。原核载体还包括噬菌体DNA如M13和其它丝状单链DNA噬菌体的衍生物。可用于酵母的载体的实例是2μ质粒。用于在哺乳动物细胞中表达的合适载体包括以下众所周知的衍生物：SV-40、腺病毒、逆转录病毒衍生的DNA序列以及衍生自功能性哺乳动物载体(如上述那些)和功能性质粒和噬菌体DNA的组合的穿梭载体。
另外的真核表达载体为本领域已知的(例如，P J.Southern&P.Berg,J.Mol.Appl.Genet,1:327-341(1982)；Subramani等人,Mol.Cell.Biol,1:854-864(1981)；Kaufinann&Sharp,"Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate  Reductase Complementary DNA Gene,"J.Mol.Biol,159:601-621(1982)；Kaufhiann&Sharp,Mol.Cell.Biol,159:601-664(1982)；Scahill等人,"Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"Proc.Nat'l Acad.Sci USA,80:4654-4659(1983)；Urlaub&Chasin,Proc.Nat'l Acad.Sci USA,77:4216-4220,(1980)，将其全部通过引用并入本文)。
可用于本申请的表达载体含有至少一个表达控制序列，其与待表达的DNA序列或片段可操作连接。将控制序列插入载体中以控制和调节克隆的DNA序列的表达。有用的表达控制序列的实例是lac系统，trp系统，tac系统，trc系统，噬菌体λ的主要操纵子和启动子区，fd外壳蛋白的控制区，酵母的糖酵解启动子，例如3-磷酸甘油酸激酶的启动子，酵母酸性磷酸酶的启动子，例如Pho5，酵母α-交配因子的启动子，以及来源于多瘤病毒、腺病毒、逆转录病毒和猿猴病毒的启动子，例如SV40的早期和晚期启动子和已知控制原核或真核细胞及其病毒或其组合的基因表达的其它序列。
在另一方面，本申请提供了一种宿主细胞，其包含所述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在另一方面，本申请提供了一种制备上述任一种纳米抗体或其抗原结合片段或多特异性分子的方法，其包括培养所述宿主细胞，以及分离所述细胞表达的纳米抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
在另一方面，本申请提供了一种制备所述免疫效应细胞的方法，其包括将编码所述CAR的核酸片段导入所述免疫效应细胞，可选地，还包括启动所述免疫效应细胞表达所述CAR。
在另一方面，本申请提供了一种药物组合物，其包含上述任一项纳米抗体或其抗原结合片段，多特异性抗体，免疫效应细胞，核酸片段，载体，宿主细胞，或上述任一项方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的载体(carrier)、稀释剂或助剂。
在一些实施方案中，所述药学上可接受的载体为不减弱免疫细胞活力以及功能、不影响抗体或其抗原结合片段与抗原特异性结合的载体，包括但不限于细胞培养基、缓冲液、生理盐水和平衡盐溶液等。缓冲液的实例包括等渗磷酸盐、醋酸盐、柠檬酸盐、硼酸盐以及碳酸盐等。在具体的实施方案中，所述药学上可接受的载体为含1％血清的磷酸盐缓冲液。
本文公开的纳米抗体或其抗原结合片段及其药物组合物能够用于治疗、改善或预防个体的肿瘤。
本文公开的纳米抗体或其抗原结合片段及其药物组合物可以任何合适的方式施用。优选地，本申请的纳米抗体或其抗原结合片段及其药物组合物通过注射(例如，皮下，静脉内，肿瘤内，动脉内，肌肉内，皮内，腹膜内或鞘内)施用。优选地，本申请的纳米抗体或其抗原结合片段及其药物组合物通过静脉内施用。对于本申请的纳米抗体或其抗原结合片段及其药物组合物，用于注射的合适的药学可接受的载体可以包括任何等张载体，例如生理盐水(含约0.90％w/v NaCl的水，含约300mOsm/L NaCl的水，或者每升水约9.0g NaCl)、NORMOSOL R电解质溶液(Abbott,Chicago,IL)、PLASMA-LYTE A(Baxter,Deerfield,IL)、含约5％葡萄糖的水或者乳酸林格氏液。在具体的实施方案中，用人血清白蛋白替换药学上可接受的载体。
具体地，所述的药物组合物还可以包含用于治疗、改善或预防个体的肿瘤的第二药剂。例如各种化疗药物，例如烷化剂化疗药物，包括但不限于环磷酰胺、异环磷酰胺、苯丙氨酸 氮芥、卡莫斯汀、洛莫斯汀、尼莫斯汀、福莫斯汀等；抗代谢类化疗药物，包括但不限于甲氨蝶呤、氟尿嘧啶、替加氟、卡莫氟、脱氧氟尿苷、卡培他滨、吉西他滨、雷替曲塞等；抗癌抗生素，包括但不限于放线菌素D、丝裂霉素、博来霉素、柔红霉素、阿霉素、表阿霉素、吡喃阿霉素等；植物类化疗药物，包括但不限于长春新碱、长春地辛、长春瑞滨、伊立替康、拓扑替康、紫杉醇、多烯紫杉醇等；杂类，包括但不限于达卡巴嗪、顺铂、卡铂、奥沙利铂、奈达铂等。
在另一方面，还提供了本申请公开的上述任一项的纳米抗体或抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物在制备预防和/或治疗肿瘤的药物中的用途；其中所述肿瘤可以为细胞表面表达MUC17的肿瘤，例如涉及消化系统的肿瘤，例如胃癌、胰腺癌和/或胃食管交界部癌。
在另一方面，本申请提供了一种预防和/或治疗肿瘤的方法，包含向有此需要的患者施用有效量的上述任一项纳米抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物；其中所述肿瘤可以为细胞表面表达MUC17的肿瘤，例如涉及消化系统的肿瘤，例如胃癌、胰腺癌和/或胃食管交界部癌。
在另一方面，本申请还提供了上述任一种纳米抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物，用于预防和/或治疗肿瘤的用途；其中所述肿瘤可以为细胞表面表达MUC17的肿瘤，例如涉及消化系统的肿瘤，例如胃癌、胰腺癌和/或胃食管交界部癌。
在另一方面，本申请提供了一种试剂盒，其包含上述任一项纳米抗体或其抗原结合片段，多特异性抗体，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物。
在另一方面，本申请提供了一种检测MUC17表达的方法，其中，在上述任一项纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使待检测样品与上述任一项纳米抗体或其抗原结合片段接触。
在另一方面，本申请提供了一种体外抑制表达MUC17细胞增殖或迁移的方法，其中，在上述任一项纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使所述细胞与上述任一项纳米抗体或其抗原结合片段接触。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请的实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1、重组蛋白和对照抗体的制备及其纯化方法
1.1重组蛋白的设计和表达
将含有编码人MUC17蛋白胞外区截短体的氨基酸序列(UniProt：Q685J3)克隆到带有His标签的pTT5载体(优宝生物，VT2202)，按照质粒抽提试剂盒方法制备质粒，并在Expi 293F细胞(Gibco，A14527)瞬转表达，获得实施例部分中所用的抗原及检测用蛋白。食蟹猴MUC17胞外区截短体蛋白的制备方法同人重组蛋白制备方法。食蟹猴MUC17序列来自于Uniprot号：A0A2K5WH09，重组蛋白的具体序列信息如下所示：
human MUC17 ECD4131-his(His标签人MUC17蛋白胞外区融合蛋白)(SEQ ID NO.1)：
Figure PCTCN2022126410-appb-000001
Cyno MUC17 ECD3577-his(His标签食蟹猴MUC17蛋白胞外区融合蛋白)(SEQ ID NO.2)：
Figure PCTCN2022126410-appb-000002
1.2 MUC17对照抗体的重组表达纯化
实施例中采用的对照抗体均来自于已公开发表的专利序列，2D11和4C11抗体序列来源于已公开发表的专利WO2019133961A1，如无特别说明，2D11和4C11对照抗体采用人IgG1+κ亚型进行重组表达。实施例中所用的纳米抗体及其人源化抗体全部采用人Fc融合形式进行重组表达。
对照抗体的表达和纯化过程如下：将抗体序列基因合成后克隆至表达载体pTT5上，然后瞬时转染Expi293F细胞(购自Gibco，A14527)，37℃摇床培养7天后收集细胞上清进行protein A抗体纯化，纯化过程参见“1.3.2Protein A亲和层析纯化对照抗体”。所得对照抗体命名为2D11-hIgG1和4C11-hIgG1。抗体具体序列信息见表1。
表1.对照抗体序列表
Figure PCTCN2022126410-appb-000003
Figure PCTCN2022126410-appb-000004
1.3重组蛋白以及对照抗体的纯化
1.3.1镍柱纯化重组蛋白
根据步骤“1.1重组蛋白的设计和表达”构建和表达相关重组蛋白后，按以下方法进行纯化：将细胞表达上清样品高速离心去除杂质，用20mM PBS+500mM NaCl溶液平衡镍柱，冲洗2-5倍柱体积。将培养上清液上样到Ni亲和层析柱(购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化，用平衡液冲洗柱子，至A280读数降至基线，后分别用含有不同浓度的咪唑梯度洗脱，收集各洗脱峰，根据SDS-PAGE胶图确定目的蛋白所在组分。收集洗脱产物，浓缩后可用凝胶层析Superdex200(GE)进一步精纯。所得到的蛋白经电泳和HPLC纯度检测合格后分装备用。通过此方案纯化得到蛋白包括人MUC17-His和猴MUC17-His。
1.3.2 Protein A亲和层析纯化对照抗体
将获得的抗体序列分别克隆到带有人Fc标签的真核表达载体pTT5上，经PEI(Polysciences，24765-1)瞬转Expi293F细胞，培养7天后，高速离心收取表达抗体的细胞培养上清。利用3-5倍柱体积的0.1M NaOH洗Protein A(博格隆，AA0273)蛋白层析柱，然后利用3-5倍柱体积的纯水清洗。利用3-5倍柱体积的1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡。细胞上清用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用3-5倍柱体积的1×PBS(pH7.4)洗涤层析柱至紫外吸收回落至基线。利用50mM柠檬酸/柠檬酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率 分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。经过protein A蛋白亲和纯化，收集从层析柱上洗脱下来的带人Fc标签的抗体，得到对应的对照抗体。
实施例2、针对MUC17蛋白的纳米抗体制备
2.1动物免疫和血清效价检测
选择2只约3周龄的骆驼，免疫前采血5ml，留作阴性对照血清。初次免疫将0.2mg实施例1制备的人MUC17蛋白与弗氏完全佐剂(购自Sigma，F5881)充分混合后颈部皮下多点注射免疫。二周后，进行第二次免疫，0.1mg人MUC17蛋白与弗式不完全佐剂混合后颈部皮下多点注射免疫，一周后取血清测定效价。酶联免疫吸附实验(ELISA)检测血清中针对人和猴MUC17蛋白的抗体效价和特异性，第四次免疫的血清效价结果如表2所示，表中的数据为OD450nm值。
表2.ELISA检测MUC17蛋白免疫后的血清抗体效价
Figure PCTCN2022126410-appb-000005
2.2纳米抗体噬菌体文库的构建
免疫结束后分别采取外周血50mL，采用淋巴细胞分离液分离PBMC。用Trizol试剂提取总RNA，按照逆转录试剂盒PrimeScript TMII 1st Strand cDNA Synthesis Kit(Takara，6210A)操作获得cDNA。采用巢式PCR扩增纳米抗体(VHH)片段。PCR产物经过纯化后，使用限制性内切酶SfiI(购自NEB，R0123L)将其连接入噬菌体展示载体pComb3Xss中，并纯化连接产物。随后将连接产物电转TG1感受态细胞，共进行了7次电击转化，电击之后立即向电击杯中加入1mL SOC培养基复苏培养1小时，共计获得7.3ml TG1细胞复苏产物。对TG1菌液梯度稀释10 3倍和10 4倍，测定纳米抗体库的转化子数目，将10 3和10 4倍稀释的TG1菌液涂布于150mm的平板上，其余菌液涂布于7块150mm的平板上。计算库容的大小分别为1.5×10 9和1.3×10 9。测序96个样品，显示文库的插入率99％左右，无重复序列，表明噬菌体文库构建成功。
2.3针对MUC17纳米抗体的淘选
本专利采用人和猴MUC17蛋白交叉淘选的方法。第一轮生物淘选，准备A、B、C三管，在A管中先加入100μL链霉亲和素偶联的Dynabeads(购自Invitrogen)及上述噬菌体抗体库，在B管中先加入100μL链霉亲和素偶联的Dynabeads。然后在三管中分别加入封闭液，即含 有20％(w/v)脱脂奶粉的PBS磷酸缓冲液，室温封闭2小时。将C管中的液体倒掉，加入A管离心后收集的上清，然后加入4μg生物素化人MUC17-His蛋白，生物素化根据试剂盒说明书(购自同仁化学，LK03)操作，室温振荡孵育2小时。并且设置对照管，仅加入未生物素化的人MUC17-His蛋白，室温振荡孵育1小时。将B管离心获得封闭后的磁珠，加入孵育后的混合液，室温振荡孵育15分钟。置于磁力架中30秒，用1mL PBST，即含有0.01％(v/v)Tween-20的封闭液洗涤10次，再用PBS缓冲液洗涤1次。洗涤后，每管加入500μl的10μg/mL胰酶，37℃孵育15分钟以洗脱与生物素化人MUC17-His蛋白结合的噬菌体。将250μl胰酶溶液加到4mL处于对数生长期的大肠杆菌TG1(购自LUCIGEN)中，37℃孵育30分钟，得到TG1的培养液。将TG1的培养液梯度稀释，涂布平板，37℃培养过夜。计算所得的与生物素化人MUC17-His蛋白结合的和对照管的克隆数，并挑选48个克隆测序。同时，将平板上的克隆用2YT培养基(购自生工，2YT培养基的配制方法为：将31g 2YT培养基粉末加入1L水中，用NaOH调至pH7.0，高压灭菌)洗涤、收集，并接种到新鲜培养基中，37℃培养至对数期。加入辅助噬菌体M13KO7(购自NEB，货号N0315S)，辅助噬菌体与大肠杆菌TG1比例为20：1，混匀，37℃静置30分钟。然后37℃振荡培养30分钟，4000rpm离心10分钟后收集细胞，加入含氨苄和卡那抗性的新鲜2YT培养基，30℃振荡培养过夜。5000rpm离心20分钟，收集上清，加入1/4上清体积的含有20％PEG6000的2.5M的NaCl溶液，冰上放置过夜。5000rpm，4℃离心30分钟，收集噬菌体沉淀，溶解在PBS缓冲液中。10000rpm离心10分钟去除残留的细胞碎片，收集上清用于下一轮的生物淘选。
第二轮淘选步骤与第一轮一致，第二轮富集与生物素化的猴MUC17-His蛋白特异性结合的VHH抗体。经过多轮的淘选，阳性噬菌体在淘选的过程中不断富集，以便筛选出特异性好、亲和力高的纳米抗体。
2.4酶联免疫方法筛选阳性噬菌体克隆
从第二、三轮的平板中挑选单克隆于96孔板培养，每孔中加入200μL含抗生素和1％葡萄糖的2YT培养基，37℃、250rpm振荡培养过夜。取10μL过夜培养物加到100μl含抗生素和0.5％葡萄糖的2YT培养基，培养至OD600为0.4-0.6，按20：1的侵染比例加入辅助噬菌体，37℃静置30分钟。然后37℃振荡培养30分钟，再加入400μl含抗生素的2YT培养基，30℃培养过夜。第二天，5000rpm，4℃离心20分钟，所得上清用于单克隆ELISA鉴定。
将人和猴MUC17蛋白用PH值为9.6的碳酸盐缓冲液稀释至终浓度为2μg/mL，按50μL/孔加入酶标孔中，4℃包被过夜。随后每孔加入50μL噬菌体培养菌液上清和1:4000稀释的辣根过氧化物酶标记的抗M13抗体(购自义翘神州，11973-MM05T-H)，洗涤之后加入TMB显色液(购自KPL，52-00-02)显色，于450nm下测光密度。挑选同时结合人和猴MUC17蛋白的阳性克隆进行FACS检测。
2.5流式细胞实验(FACS)筛选与细胞结合的噬菌体克隆
将编码猴MUC17片段的核苷酸序列克隆到pcDNA5载体(购自通用)，制备质粒并构建过表达细胞系，选择荧光强度较高的单克隆细胞系进行后续检测。构建的过表达细胞系命名为：FlpinCHO-Cyno(3597-3964)-D2。
将内源肿瘤细胞系NUGC4(南京科佰，货号：CBP60493)和FlpinCHO-Cyno(3597-3964)-D2过表达细胞株分别在T-175细胞培养瓶中扩大培养至90％汇合 度。吸尽培养基，用PBS缓冲液洗涤1次，然后用Trypsin-EDTA(购自Gibco，货号25200072)处理和收集细胞。进行细胞计数后，用PBS磷酸缓冲液洗涤细胞2次，并稀释至2×10 6个细胞每毫升，按每孔50μL加入到96孔FACS反应板中。在PBS磷酸缓冲液中加入1％(w/w)胎牛血清作为FACS缓冲液，1000rpm 4℃离心洗涤2次。每孔加入50μL噬菌体上清，冰上孵育1小时。用FACS缓冲液离心洗涤3次，每孔加入50μL 1：1000稀释的抗M13抗体(义翘神州，货号11973-MM05T)，冰上孵育1小时，用FACS缓冲液离心洗涤3次，每孔加入荧光(Alexa 647)标记的二抗(Jackson Immuno,货号115-605-003)，冰上孵育1小时。用FACS缓冲液离心洗涤3次。用100μL的FACS缓冲液悬浮细胞，FACS上机检测和分析结果。
经过多轮的优化和筛选，共获得5个能够同时识别人和猴MUC17的阳性克隆，分别命名为Lab315、Lab316、Lab320、Lab331和Lab332。其序列的CDRs分别用KABAT、Chothia或IMGT软件分析，对应的序列信息如下表3-表4所示，其中表3示出5个纳米抗体分子氨基酸序列，表4示出5个纳米抗体分子CDRs的IMGT、Kabat和Chothia分析结果。
表3.抗MUC17纳米抗体的氨基酸具体序列信息
Figure PCTCN2022126410-appb-000006
表4.IMGT、KABAT和Chothia软件分析MUC17纳米抗体的CDRs具体序列信息
Figure PCTCN2022126410-appb-000007
Figure PCTCN2022126410-appb-000008
实施例3、重组纳米抗体表达纯化及亲和力检测
3.1重组纳米抗体的表达纯化
将获得的纳米抗体序列分别克隆到带有Fc标签的真核表达载体pTT5上，经PEI瞬转Expi293F细胞(购自Gibco,A14527)，培养7天后，高速离心收取表达抗体的细胞培养上清。并按实施例1.3.2所述纯化方法纯化抗体，得到对应的重组纳米抗体，分别命名为Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc。运用SEC-HPLC方法对纯化得到的抗体进行纯度检测，检测合格后分装备用。
3.2酶联免疫吸附实验(ELISA)检测重组抗体与人MUC17-his蛋白的结合
人MUC17-his蛋白用PBS稀释到终浓度2μg/mL，然后以50μl每孔加到96孔ELISA板，4℃孵育过夜。第二天用PBST洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入100nM为起始浓度，3倍梯度稀释的重组抗体，阳性及阴性对照抗体50μl每孔。37℃孵育1小时后，用PBST洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Merck，货号：AP113P)，37℃孵育1小时后，用PBST洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD 450nm数值，重组抗体Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc与人MUC17蛋白的 结合活性如图1A-1B所示，说明纯化后的抗体与人MUC17-his蛋白均有效结合。
3.3酶联免疫吸附实验(ELISA)检测重组抗体与猴MUC17-his的结合
将猴MUC17-his蛋白按照实施例3.2的方法进行ELISA检测与数据分析。分析结果如图2A-2B所示，重组抗体Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc与猴MUC17-his蛋白有较好的结合活性。
3.4流式细胞实验(FACS)检测重组抗体与人MUC17的结合
将内源肿瘤细胞NUGC4在T-175培养瓶中扩大培养至对数生长期，吸除培养基，用PBS缓冲液洗涤2次，用胰酶消化细胞，然后用完全培养基终止消化，并吹打细胞至单细胞悬液。细胞计数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2×10 6细胞每毫升，按每孔100μl加入到96孔FACS反应板中，离心，弃掉上清，加入待测抗体样品(100nM为起始浓度，3倍梯度稀释)每孔50μl，与细胞混匀，4℃孵育1小时。用PBS缓冲液离心洗涤3次，每孔加入50μl Alexa
Figure PCTCN2022126410-appb-000009
647 AffiniPure Goat Anti-Human IgG,Fcγfragment specific标记的二抗(购自Jackson，货号：109-605-098)，4℃孵育1小时。用PBS缓冲液离心洗涤3次，100μl PBS重选后用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(FlowJo)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50。如图3A-3B所示，重组抗体Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc均可特异性结合NUGC-4细胞。
3.5流式细胞实验(FACS)检测重组抗体与猴MUC17的结合
将FlpinCHO-Cyno(3597-3964)-D2重组细胞按照实施例3.4的方法进行FACS检测与数据分析。结果如图4A-4B所示，重组抗体Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc与FlpinCHO-Cyno(3597-3964)-D2细胞能够有效结合。
3.6重组抗体的亲和力测定
应用BIAcore 8K仪器，采用Protein A捕获法检测抗体与抗原的结合强度。首先，应用氨基偶联法将Protein A固定到CM4芯片(购自GE，BR-1005-34)上，根据Amine Coupling Kit试剂盒(购自GE，BR100633)的指导，以HBS-EP+pH7.4为流动相，将NHS和EDC混合后，活化芯片约600秒，用10mM乙酸钠pH4.5将Protein A稀释至50μg/mL，注射600s，最后用乙醇胺对剩余的活化位点进行封闭。然后，采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5完成芯片再生，其中流动相为HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)，流速30μL/min，再生时间为30s，检测温度为25℃；最后，根据1:1binding模型，对数据进行分析，拟合抗体抗原结合动力学参数，包括结合速率常数Ka、解离速率常数Kd、平衡解离常数KD、最大结合信号Rmax。重组抗体Lab315-huFc、Lab316-huFc、Lab320-huFc、Lab331-huFc和Lab332-huFc与人MUC17蛋白的结合速率(Ka)、解离速率(Kd)及结合亲和力(KD)如表5所示。
表5.SPR(Biacore)检测重组抗体与人MUC17蛋白的亲和力
Figure PCTCN2022126410-appb-000010
Figure PCTCN2022126410-appb-000011
实施例4、纳米抗体人源化
4.1纳米抗体的人源化设计
通过比对IMGT(http://imgt.cines.fr)数据库中人类抗体可变区种系基因序列，分别挑选与纳米抗体同源性高的重链可变区种系基因作为模板，将纳米抗体的基于IMGT或KABAT命名方法的CDRs序列分别移植到相应的人源模板中，形成次序为“FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4”的可变区序列。
Lab315抗体的CDR序列基于IMGT规则定义，其人源化模板为IGHV3-9*01和IGHJ3*01，Lab315抗体在人源化过程中重新命名为SCR-7724，CDR移植后命名为SCR-7724-001-H。
Lab316抗体的CDR序列基于Kabat规则定义，其人源化模板为IGHV3-7*01和IGHJ3*01，Lab316抗体在人源化过程中重新命名为SCR-7782，CDR移植后命名为SCR-7782-001-H。
Lab320抗体的CDR序列基于Kabat规则定义，其人源化模板为IGHV3-7*01和IGHJ3*01，Lab320抗体在人源化过程中重新命名为SCR-7783，CDR移植后命名为SCR-7783-001-H。
Lab331抗体的CDR序列基于IMGT规则定义，其人源化模板为IGHV3-30*02和IGHJ3*01，Lab331抗体在人源化过程中重新命名为SCR-7784，CDR移植后命名为SCR-7784-001-H。
Lab332抗体的CDR序列基于Kabat规则定义，其人源化模板为IGHV3-30*02和IGHJ3*01，Lab332抗体在人源化过程中重新命名为SCR-7785，CDR移植后命名为SCR-7785-001-H。
将抗体SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785的CDRs分别移植到其人源化模板中，即获得人源化抗体。其人源化模板的氨基酸序列以及CDR graft后的人源化抗体序列见表6。
表6.人源化模板及人源化抗体的氨基酸具体序列信息
Figure PCTCN2022126410-appb-000012
Figure PCTCN2022126410-appb-000013
4.2 SCR-7724的人源化抗体回复突变设计
将SCR-7724人源化抗体的FR区序列中关键氨基酸进行回复突变为骆驼抗体对应的氨基酸，以保证原有的亲和力，具体回复突变后的突变点详情(回复突变点以自然顺序编号)及具体氨基酸序列见表7-8。
表7.SCR-7724人源化抗体回复突变的突变点情况
Figure PCTCN2022126410-appb-000014
注：Graft(IGHV3-9*01)代表将纳米抗体CDR植入人种系FR区序列；H34G表示将Graft(IGHV3-9*01)的第34位H突变为G，其它依此类推。
表8.SCR-7724人源化抗体回复突变的氨基酸序列
Figure PCTCN2022126410-appb-000015
Figure PCTCN2022126410-appb-000016
4.3 SCR-7782的人源化抗体回复突变设计
将SCR-7782人源化抗体的FR区序列中关键氨基酸进行回复突变为骆驼抗体对应的氨基酸，以保证原有的亲和力，具体回复突变后的突变点详情(回复突变点以自然顺序编号)及具体氨基酸序列见表9-10。
表9.SCR-7782人源化抗体回复突变的突变点情况
Figure PCTCN2022126410-appb-000017
注：Graft(IGHV3-7*01)代表将纳米抗体CDR植入人种系FR区序列；V37F表示将Graft(IGHV3-7*01)的第37位V突变为F，其它依此类推。
表10.SCR-7782人源化抗体回复突变的氨基酸序列
Figure PCTCN2022126410-appb-000018
Figure PCTCN2022126410-appb-000019
4.4 SCR-7783的人源化抗体回复突变设计
将SCR-7783人源化抗体的FR区序列中关键氨基酸进行回复突变为骆驼抗体对应的氨基酸，以保证原有的亲和力，具体回复突变后的突变点详情(回复突变点以自然顺序编号)及具体氨基酸序列见表11-12。
表11.SCR-7783人源化抗体回复突变的突变点情况
Figure PCTCN2022126410-appb-000020
注：Graft(IGHV3-7*01)代表将纳米抗体CDR植入人种系FR区序列；V37F表示将Graft(IGHV3-7*01)的第37位V突变为F，其它依此类推。
表12.SCR-7783人源化抗体回复突变的氨基酸序列
Figure PCTCN2022126410-appb-000021
Figure PCTCN2022126410-appb-000022
4.5 SCR-7784的人源化抗体回复突变设计
将SCR-7784人源化抗体的FR区序列中关键氨基酸进行回复突变为骆驼抗体对应的氨基酸，以保证原有的亲和力，具体回复突变后的突变点详情(回复突变点以自然顺序编号)及具体氨基酸序列见表13-14。
表13.SCR-7784人源化抗体回复突变的突变点情况
Figure PCTCN2022126410-appb-000023
注：Graft(IGHV3-30*02)代表将纳米抗体CDR植入人种系FR区序列；H35A表示将Graft(IGHV3-30*02)的第35位H变为A其它依此类推。
表14.SCR-7784人源化抗体回复突变的氨基酸序列
Figure PCTCN2022126410-appb-000024
Figure PCTCN2022126410-appb-000025
4.6 SCR-7785的人源化抗体回复突变设计
SCR-7785抗体的CDR序列基于Kabat规则定义。将SCR-7785人源化抗体的FR区序列中关键氨基酸进行回复突变为骆驼抗体对应的氨基酸，以保证原有的亲和力，具体回复突变后的突变点详情(回复突变点以自然顺序编号)及具体氨基酸序列见表15-16。
表15.SCR-7785人源化抗体回复突变的突变点情况
Figure PCTCN2022126410-appb-000026
注：Graft(IGHV3-30*02)代表将骆驼抗体CDR植入人种系FR区序列；V37F表示将Graft(IGHV3-30*02)的第37位V突变为F，其它依此类推。
表16.SCR-7785人源化抗体回复突变的氨基酸序列
Figure PCTCN2022126410-appb-000027
Figure PCTCN2022126410-appb-000028
实施例5、人源化纳米抗体的表达纯化及鉴定
5.1人源化纳米抗体的表达纯化
将人源化抗体可变区序列基因合成后克隆到带有人铰链区和Fc恒定区序列的pTT5载体，形成VHH-huFc(C220S)表达顺序，并制备质粒。抗体质粒经PEI瞬时转染到Expi293F细胞，培养7天后收集上清，按实施例1.3.2方式经protein A纯化制备抗体。
5.2酶联免疫吸附实验(ELISA)检测人源化抗体与人MUC17-his蛋白的结合
参见实施例3.2的方法步骤检测人源化抗体与人MUC17-his蛋白的结合活性。实验结果如图5A-5E所示，人源化抗体SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785均维持了重组抗体的结合能力，与人MUC17蛋白具有很好的结合活性。
5.3 ELISA检测人源化抗体与猴MUC17-his蛋白的结合活性
为检测人源化抗体与猴MUC17-his蛋白的结合活性，将猴MUC17-his蛋白用PBS稀释到终浓度2μg/mL，按照实施例3.3的方法进行ELISA检测与数据分析。结果如图6A-6E所示，人源化抗体SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785与猴MUC17蛋白具有交叉结合活性，维持了重组抗体的结合能力。
5.4流式细胞实验(FACS)检测人源化抗体与内源肿瘤细胞NUGC4的结合
检测细胞和待测抗体的准备以及检测方法参照实施例3.4。检测结果如图7A-7E所示，人源化抗体均可与内源细胞NUGC4有较好的结合活性，维持了人源化前对应重组抗体的结合能力。
5.5流式细胞实验(FACS)检测人源化抗体与猴MUC17过表达细胞的结合
猴MUC17过表达细胞FlpinCHO-Cyno(3597-3964)-D2的准备以及FACS检测方法参照实施例3.4和3.5。实验结果如图8A-8E所示，本实施例中的人源化抗体SCR-7724、SCR-7782、SCR-7783、SCR-7784和SCR-7785均可与FlpinCHO-Cyno(3597-3964)-D2重组细胞有较好的交叉结合活性，维持了重组抗体相当的结合能力。
实施例6、人源化抗体的亲和力测定
将最终获得的多种人源化抗体表达纯化后，按照实施例3.6的方法测定人源化抗体与人MUC17蛋白的亲和力，具体亲和力数值见表17。
表17.SPR(biacore)检测人源化抗体与人MUC17蛋白的亲和力
Figure PCTCN2022126410-appb-000029
Figure PCTCN2022126410-appb-000030
虽然，上文中已经用一般性说明及具体实施方案对本申请的技术方案作了详尽的描述，但在这些技术方案的基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本申请精神的基础上所做的这些修改或改进，均属于本申请要求保护的范围。

Claims (27)

  1. 一种特异性结合MUC17的纳米抗体或其抗原结合片段，其中，所述纳米抗体或其抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别选自SEQ ID NO.8～12、62～66、67～98中任一项所示的VHH的CDR1、CDR2和CDR3。
  2. 根据权利要求1所述的纳米抗体或其抗原结合片段，其中所述CDR1、CDR2和CDR3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定，例如，所述CDR1包含SEQ ID NO.13、16、19、22、25、28、31、34、37、40、43、46、49、52或55所示的氨基酸序列；所述CDR2包含SEQ ID NO.14、17、20、23、26、29、32、35、38、41、44、47、50、53或56所示的氨基酸序列；所述CDR3包含SEQ ID NO.15、18、21、24、27、30、33、36、39、42、45、48、51、54或57所示的氨基酸序列。
  3. 根据权利要求1或2任一项所述的纳米抗体或其抗原结合片段，其中SEQ ID NO.8、62、67～71所示的VHH的CDR1、CDR2和CDR3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：13～15、SEQ ID NO：28～30或SEQ ID NO：43～45所示的氨基酸序列；
    SEQ ID NO.9、63、72～77所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：16～18、SEQ ID NO：31～33或SEQ ID NO：46～48所示的氨基酸序列；
    SEQ ID NO.10、64、78～84所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：19～21、SEQ ID NO：34～36或SEQ ID NO：49～51所示的氨基酸序列；
    SEQ ID NO.11、65、85～91所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：22～24、SEQ ID NO：37～39或SEQ ID NO：52～54所示的氨基酸序列；
    SEQ ID NO.12、66、92～98所示的VHH的CDR1～3按照IMGT、Kabat或Chothia编号系统，并且具有如SEQ ID NO：25～27、SEQ ID NO：40～42或SEQ ID NO：55～57所示的氨基酸序列。
  4. 根据权利要求1-3任一项所述的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段包含与所述CDR1、CDR2和CDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDR1、CDR2和CDR3序列，优选地，所述替换为保守氨基酸的替换。
  5. 根据权利要求1-4任一项所述的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段包含SEQ ID NO.8～12、62～66、67～98任一项所示的VHH，或者与SEQ ID NO.8～12、62～66、67～98任一项所示的VHH具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性或至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的VHH序列；所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  6. 根据权利要求5所述的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段包含与SEQ ID NO：62所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，H34G，V36F，G43E，L44R，W46G，S48A，G57I，R70K，Y93S，W114R或M119Q；优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，G57I，R70K，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，R70K，Y93S和W114R突变；更优选地，至少具有H34G，V36F，G43E，L44R，W46G，S48A，R70K，Y93S，W114R和M119Q突变；
    所述抗体或其抗原结合片段包含与SEQ ID NO：63所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，G26D，F27N，V37F，G44E，L45R，W47G，R71Q，V92M，R97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有V37F，G44E，L45R，W47G，R71Q和R97突变；更优选地，至少具有F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，G26D，F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，V5Q，E6A，G26D，F27N，V37F，G44E，L45R，W47G，R71Q和R97A突变；更优选地，至少具有E1D，G26D，F27N，V37F，G44E，L45R，W47G，R71Q，V92M，R97A和M118Q突变；
    所述抗体或其抗原结合片段包含与SEQ ID NO：64所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S，Y79W，V92M，R97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有T28P，F29S，V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S，Y79W和R97A突变；更优选地，至少具有E1D，V5Q，E6A，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S和R97A突变；更优选地，至少具有E1D，T28P，F29S，V37F，G44E，L45R，W47G，K75L，N76S，V92M，R97A和M118Q突变；
    所述抗体或其抗原结合片段包含与SEQ ID NO：65所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，E1D，V5Q，E6A，H35A，V37F，G44E，L45R，W47V，F68L，R72H，S75A，V93M或M119Q；优选地，至少具有H35A，V37F，G44E，L45R和W47V突变；更优选地，至少具有H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，R72H和S75A突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，F68L，R72H和S75A突变；更优选地，至少具有E1D，V5Q，E6A，H35A，V37F，G44E，L45R，W47V和R72H突变；更优选地，至少具有E1D，H35A，V37F，G44E，L45R，W47V，R72H，V93M和M119Q突变；
    或所述抗体或其抗原结合片段包含与SEQ ID NO：66所示的VHH的框架区相比，至少具有选自下组的突变的框架区序列：按自然顺序编号，V5Q，E6A，F27D，V37F，A40R，G44E，L45R，W47A，R71Q，S74T，V92M，K97A或M118Q；优选地，至少具有V37F，G44E，L45R，W47A和K97A突变；更优选地，至少具有V37F，G44E，L45R，W47A，R71Q和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q，S74T和K97A突变；更优选地，至少具有F27D，V37F，G44E，L45R，W47A，R71Q，V92M，K97A和M118Q突变；更优选地，至少具有F27D，V37F，A40R，G44E，L45R，W47A，R71Q，V92M，K97A和M118Q突变；更优选地，至少具有V5Q，E6A，F27D，V37F，G44E，L45R，W47A，R71Q和K97A突变。
  7. 根据权利要求1～6任一项所述的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段与人MUC17蛋白和/或猴MUC17蛋白特异性结合；优选地，其与人MUC17蛋白和/或猴MUC17蛋白结合的KD优于1.00E-7M。
  8. 根据权利要求1～7任一项所述的纳米抗体或其抗原结合片段，其中所述纳米抗体或其抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  9. 根据权利要求1～8任一项的纳米抗体或其抗原结合片段，其包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区选自IgG、IgM、IgA、IgE或IgD，所述IgG选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述纳米抗体或其抗原结合片段为重链抗体。
  10. 根据权利要求1～9任一项的纳米抗体或其抗原结合片段，其还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  11. 一种多特异性分子，其包含权利要求1～10中任一项的纳米抗体或其抗原结合片段；优选地，所述多特异性分子进一步包含特异性结合MUC17以外的抗原或结合与权利要求1～10任一项所述的纳米抗体或其抗原结合片段不同的MUC17表位的纳米抗体或其抗原结合片段。
  12. 根据权利要求11的多特异性分子，其中，所述MUC17以外的抗原为T细胞、B细胞、自然杀伤细胞、树突状细胞、巨噬细胞、单核细胞或嗜中性细胞表面上的抗原；优选地，所述MUC17以外的抗原选自：CD96、PD-1、PD-L1、PD-L2、OX40、OX40L、LAG-3、TIM3、VISTA、CD3、CD3γ、CD3δ、CD3ε、CD3ζ、CD27、CD28、CD28H、CD16、CD16A、CD32B、VEGF、NKG2D、NKp30、NKp46、NKp44、CD19、CD20、CD40、CD47、4-1BB、ICOS、OX40、EGFR、EGFRvIII、TNF-alpha、CD33、HER2、HER3、HAS、CD5、CD27、EphA2、EpCAM、MUC1、MUC16、CEA、Claudin18.2、叶酸受体、Claudin6、WT1、NY-ESO-1、MAGE3、ASGPR1、TGFβ-trap、IL-2、IL-15、IL-21、IL-18或CDH16；
    优选地，所述多特异性分子为双特异性、三特异性或四特异性，更优选地，所述多特异性分子为二价、四价或六价。
  13. 根据权利要求11或12的多特异性分子，其为串联scFv、双功能抗体(Db)、单链双功能抗体(scDb)、双重亲和力再靶向(DART)抗体、F(ab')2、双重可变域(DVD)抗体、臼包杵(KiH)抗体、对接及锁定(DNL)抗体、化学交联抗体、杂多聚纳米抗体或异结合物抗体。
  14. 一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1～10任一项所述的纳米抗体或其抗原结合片段。
  15. 一种免疫效应细胞，其表达权利要求14所述的嵌合抗原受体，或包含编码权利要求14所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  16. 一种分离的核酸片段，其编码权利要求1～10任一项所述的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述多特异性分子，或权利要求14所述的嵌合抗原受体。
  17. 一种载体(vector)，其包含权利要求16所述的核酸片段。
  18. 一种宿主细胞，其包含权利要求17所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
  19. 一种制备权利要求1～10任一项纳米抗体或其抗原结合片段或权利要求11～13任一项所述的多特异性分子的方法，其包括培养权利要求18所述的细胞，以及分离所述细胞表达的纳米抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
  20. 一种制备权利要求15所述免疫效应细胞的方法，其包括将编码权利要求14所述的CAR的核酸片段导入所述免疫效应细胞，可选地，还包括启动所述免疫效应细胞表达权利要求14所述的CAR。
  21. 一种药物组合物，其包含权利要求1～10任一项所述的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述的多特异性抗体，或权利要求15所述的免疫效应细胞，或权利要求16的核酸片段，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19～20任一项所述的方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的载体、稀释剂或助剂。
  22. 权利要求1～10任一项的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述的多特异性分子，或权利要求15所述的免疫效应细胞，或权利要求16所述的核酸片段，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19～20任一项所述的方法制备获得的产品；或权利要求21所述的药物组合物在制备预防和/或治疗肿瘤的药物中的用途；任选地，所述肿瘤为细胞表面表达MUC17的肿瘤，例如胃癌、胰腺癌、胃食管交界部癌。
  23. 一种预防和/或治疗肿瘤的方法，包括向有此需要的患者施用有效量的权利要求1～10任一项的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述的多特异性分子，或权利要求15所述的免疫效应细胞，或权利要求16所述的核酸片段，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19～20任一项所述的方法制备获得的产品；或权 利要求21所述的药物组合物；任选地，所述肿瘤为细胞表面表达MUC17的肿瘤，例如胃癌、胰腺癌、胃食管交界部癌。
  24. 权利要求1～10任一项的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述的多特异性分子，或权利要求15所述的免疫效应细胞，或权利要求16所述的核酸片段，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19～20任一项所述的方法制备获得的产品；或权利要求21所述的药物组合物，用于预防和/或治疗肿瘤的用途；任选地，所述肿瘤为细胞表面表达MUC17的肿瘤，例如胃癌、胰腺癌和胃食管交界部癌。
  25. 一种试剂盒，其包含权利要求1～10任一项所述的纳米抗体或其抗原结合片段，或权利要求11～13任一项所述的多特异性抗体，或权利要求15所述的免疫效应细胞，或权利要求16所述的核酸片段，或权利要求17所述的载体，或权利要求18所述的宿主细胞，或权利要求19～20任一项所述的方法制备获得的产品，或权利要求21所述的药物组合物。
  26. 一种检测MUC17表达的方法，其中，在权利要求1～10任一项所述的纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使待检测样品与权利要求1～10任一项所述的纳米抗体或其抗原结合片段接触。
  27. 一种体外抑制表达MUC17的细胞增殖或迁移的方法，其中，在权利要求1～10任一项所述的纳米抗体或其抗原结合片段与MUC17之间能够形成复合物的条件下，使所述细胞与权利要求1～10任一项所述的纳米抗体或其抗原结合片段接触。
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