WO2024104431A1 - FRα-TARGETING ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, AND USE THEREOF - Google Patents

FRα-TARGETING ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, AND USE THEREOF Download PDF

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WO2024104431A1
WO2024104431A1 PCT/CN2023/132065 CN2023132065W WO2024104431A1 WO 2024104431 A1 WO2024104431 A1 WO 2024104431A1 CN 2023132065 W CN2023132065 W CN 2023132065W WO 2024104431 A1 WO2024104431 A1 WO 2024104431A1
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antibody
seq
frα
determining regions
antigen
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PCT/CN2023/132065
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French (fr)
Chinese (zh)
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毕建军
桂勋
吴建
徐晓红
胡蓉蓉
王晋
蔺利娟
任红媛
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迈威(上海)生物科技股份有限公司
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Publication of WO2024104431A1 publication Critical patent/WO2024104431A1/en

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    • 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
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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Definitions

  • the present invention belongs to the field of antibodies, and specifically relates to a FR ⁇ binding molecule, in particular an antibody and a fragment thereof that specifically recognizes FR ⁇ .
  • the present invention also relates to a nucleic acid or host cell containing such an antibody or a fragment thereof, a drug containing such an antibody or a fragment thereof, and a treatment and diagnosis method or use using these antibodies and fragments.
  • the invention discloses an antibody or an antigen binding fragment thereof targeting FR ⁇ and application thereof.
  • the present invention also provides methods for blocking FR ⁇ -mediated signaling pathways in subjects using the antibodies of the present invention, as well as methods for preventing or treating FR ⁇ -related diseases, such as immune system diseases (eg, autoimmune diseases or inflammation).
  • FR ⁇ -related diseases such as immune system diseases (eg, autoimmune diseases or inflammation).
  • Figure 1 shows the mouse serum FR ⁇ antibody titer (absorbance measured at 650 nM);
  • FIG3 shows FACS detection of the binding of hybridoma supernatants to CHO cells overexpressing hFR ⁇ (CHO-hFOLR1);
  • Figure 4 shows the FACS detection of the hybridoma supernatant binding to CHO cells overexpressing cyno FR ⁇ (CHO-cynoFOLR1);
  • Figures 10-13 show the binding ability of different anti-FR ⁇ chimeric antibodies to different cells, wherein antibody number -xiIgG refers to a chimeric antibody having a heavy chain constant region of IgG1 and a ⁇ subclass light chain constant region as prepared in Example 2.6, and its variable region refers to the variable region corresponding to the "antibody number" in Table 1; MIRV: Mirvecuximab, which serves as a positive control (PC); NC: negative control, IgG1.
  • Figures 14-21 show the in vitro cell killing results (RLU curves and IC50 values) of ADCs of different anti-FR ⁇ chimeric antibodies, wherein Antibody No.-ADC refers to the ADC of a chimeric antibody having a heavy chain constant region of IgG1 and a ⁇ subclass light chain constant region and vcMMAE as prepared in Example 2.6, MIRV-ADC refers to the ADC molecule MIRV-vcMMAE of MIRV, and NC-ADC refers to the ADC of IgG1 and vcMMAE.
  • Antibody No.-ADC refers to the ADC of a chimeric antibody having a heavy chain constant region of IgG1 and a ⁇ subclass light chain constant region and vcMMAE as prepared in Example 2.6
  • MIRV-ADC refers to the ADC molecule MIRV-vcMMAE of MIRV
  • NC-ADC refers to the ADC of IgG1 and vcMMAE
  • Figures 22-23 show the Octet-assessed binding kinetics results of the humanized antibodies hz2F22-H1L1, hz2F22-H1L0, hzPha3-H0L0, hzPha3-H1L2, hzPha3-H1L1, hzPha3-H0L1 and hzPha3-H1L0 prepared as in Example 3.1 and the chimeric antibodies Pha3-xiIgG and 2F22-xiIgG with human FR ⁇ .
  • Figures 26-27 show the humanized antibodies hz2F22-H1L1, hz2F22-H1L0, hzPha3-H0L0, hzPha3-H1L2, hzPha3-H1L1, hzPha3-H0L1 and hzPha3-H1L0 and chimeric antibodies prepared as in Example 3.1
  • Figure 37 shows the effects of hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, and Mirve-MMAE1 mg/Kg administration groups on the growth of human cervical cancer KB subcutaneous transplanted tumors.
  • Figure 38 shows the effects of hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, and Mirve-MMAE 3 mg/Kg administration groups on the growth of human cervical cancer KB subcutaneous transplanted tumors.
  • the term “comprising” or “including” means including the elements, integers or steps described, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise specified, it also covers the combination of the elements, integers or steps described.
  • an antibody variable region “comprising” a specific sequence when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.
  • FR ⁇ refers to any natural FR ⁇ polypeptide (e.g., human FR ⁇ polypeptide) or variant thereof.
  • FR ⁇ encompasses "full-length” unprocessed FR ⁇ polypeptides as well as any form of FR ⁇ polypeptides produced by processing within a cell.
  • the term also encompasses naturally occurring variants of FR ⁇ , such as those encoded by splice variants and allelic variants.
  • the FR ⁇ polypeptides described herein can be isolated from a variety of sources, such as from human tissue or from another source, such as rhesus monkeys, or prepared by recombinant or synthetic methods.
  • the -nucleoside of the human FR ⁇ protein The acid sequence is in one embodiment of the present invention, the nucleotide sequence of the rhesus monkey FR ⁇ protein is As shown in accession number NM_001194647.3.
  • anti-FR ⁇ antibody refers to an antibody that is capable of binding to (primate, such as human or rhesus monkey) FR ⁇ or a fragment thereof with sufficient affinity.
  • the terms “whole antibody” or “full-length antibody” are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules.
  • the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds.
  • the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.
  • the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3.
  • the full-length antibody light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region, wherein the light chain constant region consists of one domain CL.
  • VL light chain variable region
  • Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the term "antibody fragment” includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.
  • antigen-binding fragment of an antibody is a molecule different from a full-length antibody, which contains a portion of a full-length antibody, but which can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody, sc(Fv) 2 .
  • Fab fragments can be obtained by digesting full-length antibodies with papain.
  • digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and is a divalent antibody fragment.
  • F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer.
  • a Fab' monomer is essentially a Fab fragment with a hinge region.
  • the Fv fragment consists of the VL and VH domains of a single arm of an antibody.
  • the two domains VL and VH of the Fv fragment can be encoded by independent genes, but recombinant methods can also be used to connect the two domains using synthetic connecting peptides to produce them as a single protein chain, and in the single protein chain, the VL region and the VH region are paired to form a single-chain Fv (scFv).
  • sc(Fv)2 is a small antibody in which two VH and two VL are connected by a linker to form a single chain.
  • Fab fragment or “Fab” is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from N-terminus to C-terminus, and the other polypeptide chain comprises VL and another constant region selected from CL and CH1 from N-terminus to C-terminus, wherein the VH domain and the VL domain pair to form an antigen binding site.
  • the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as a "Fab heavy chain”; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as a "Fab light chain”.
  • Fc region or "Fc domain” herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Diabodies are small bivalent antibodies constructed by gene fusion, for example, dimers consisting of two polypeptide chains.
  • the VL and VH domains of each polypeptide chain of a diabody are bound by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region fragments.
  • Diabodies generally have two antigen binding sites.
  • CDR region is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (Kabat et al., Sequen ces of Proteins of Immunological Interest, 4th ed., U.S.
  • CDR or “CDR sequence” encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDR of the heavy chain variable region of the antibody of the present invention is determined according to Chothia. In some embodiments, the CDR of the heavy chain variable region of the antibody of the present invention is determined according to Kabat. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the longest combination of Chothia and Kabat (hereinafter shown as "Chothia & Kabat").
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment schemes are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment scheme rules or combinations).
  • chimeric antibody is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and/or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.
  • a “humanized antibody” is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the rest of the antibody with their human counterparts (i.e., replacing the constant region and the portion of the variable region that is not involved in binding with the corresponding portion of a human antibody).
  • a non-human antibody e.g., a mouse monoclonal antibody
  • the terms "anti,” “binding,” or “specific binding” mean that the binding is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).
  • an “immunoconjugate” is an antibody conjugated to one or more other substances (eg, macromolecular substances, radioactive elements, cytotoxic agents, etc.).
  • “Individuals” or “subjects” include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
  • an effective amount refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses administered to a patient, produces the desired effect in a patient in need of treatment or prevention.
  • a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%, relative to an untreated subject.
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity screened or selected in the initially transformed cells.
  • Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells.
  • Host cells include cultured cells, and also include cells inside transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused.
  • the label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label.
  • the term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
  • an “isolated” antibody or molecule is one that has been separated from a component of its natural environment.
  • the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed phase HPLC.
  • the "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the present invention contemplates variants of the antibody molecules of the present invention, which have a considerable degree of identity, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein.
  • the variants may comprise conservative changes.
  • “conservative changes” include replacement, deletion or addition of polypeptide sequences, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the term "conservative sequence change” is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence.
  • a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.
  • therapeutic agent encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in treating cancer or immune system disorders.
  • small molecule drug refers to low molecular weight organic compounds that can regulate biological processes.
  • Small molecules are defined as molecules with a molecular weight less than 10kD, usually less than 2kD, and preferably less than 10kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.
  • immunomodulator refers to a natural or synthetic agent or drug that inhibits or modulates an immune response.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressants.
  • the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • pharmaceutical excipient refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.
  • adjuvant eg, Freund's adjuvant (complete and incomplete)
  • excipient eg, carrier, stabilizer, or the like
  • composition refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • drug combination or combination product refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit/test kit, a pharmaceutical composition.
  • non-fixed combination means that the active ingredients (e.g., (i) antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient.
  • fixed combination means that two or more active agents are administered to a patient simultaneously in the form of a single entity.
  • the dosage and/or time interval of the two or more active agents are selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition.
  • Each component can be in a separate formulation, which can be the same or different.
  • the term "combination therapy” refers to the administration of two or more therapeutic agents or treatments to treat diseases described herein.
  • This administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients.
  • this administration includes the co-administration of each active ingredient in a variety of or in separate containers (e.g., tablets, capsules, powders, and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dose before administration.
  • this administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.
  • prevention includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced.
  • expression vector refers to a vector comprising a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including those incorporated into recombinant polynucleotides.
  • the invention relates to cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) containing nucleic acids.
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • tissue or cell sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • an anti-FR ⁇ antibody or fragment thereof of the invention binds primate FR ⁇ (e.g., human FR ⁇ or rhesus monkey FR ⁇ ) with high affinity, e.g., binds to FR ⁇ (e.g., human FR ⁇ ) with an equilibrium dissociation constant ( KD ) of less than about 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM , 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, or 0.08 nM, or within a range of about 0.01 nM or 0.05 nM to any of the above values, or within a range of any of the above values.
  • KD
  • the antibodies or fragments thereof of the present invention are capable of inducing endocytosis of the antibodies or fragments thereof or molecules comprising the antibodies or fragments thereof by FR-positive cells, preferably with an endocytosis efficiency equivalent to or better than known anti-FR antibodies (e.g., MIRV).
  • molecules comprising antibodies of the present invention or fragments thereof have cell (eg, tumor cell, eg, FR cell-positive cell, eg, FR cell-positive tumor cell) killing ability.
  • cell eg, tumor cell, eg, FR cell-positive cell, eg, FR cell-positive tumor cell
  • the antibodies or fragments thereof of the present invention are capable of preventing or treating FR ⁇ -related diseases, such as tumors, such as solid tumors, such as epithelial cell cancers, such as lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumors, and endometrial cancer.
  • FR ⁇ -related diseases such as tumors, such as solid tumors, such as epithelial cell cancers, such as lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumors, and endometrial cancer.
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
  • HCDRs complementarity determining regions
  • the anti-FR ⁇ antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region.
  • LCDRs complementarity determining regions
  • an anti-FR ⁇ antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL). In some aspects, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, such as HCDR1, HCDR2, and HCDR3 determined by the Kabat and/or Chothia schemes.
  • HCDRs complementary determining regions
  • the light chain variable region comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3, such as HCDR1, HCDR2 and HCDR3 determined by the Kabat and/or Chothia scheme (e.g., the Chothia & Kabat scheme).
  • LCDRs complementarity determining regions
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region and a light chain constant region.
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises an antibody heavy chain (HC). In some embodiments, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain (LC). In some embodiments, the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain.
  • the antibody heavy chain of the present invention comprises an antibody heavy chain variable region and an antibody heavy chain constant region.
  • the antibody light chain of the present invention comprises an antibody light chain variable region and an antibody light chain constant region.
  • the heavy chain variable region of the invention is a heavy chain variable region of the invention.
  • (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116; or
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116; or
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116, and preferably, the amino acid changes do not occur in the CDR regions.
  • the light chain variable region of the invention is a light chain variable region of the invention
  • (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51; or
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51; or
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51, and preferably, the amino acid changes do not occur in the CDR regions.
  • the three complementarity determining regions (HCDRs) from the heavy chain variable region of the present invention, HCDR1, HCDR2 and HCDR3 are selected from
  • HCDR regions (iv) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to the sequence of any one of (i).
  • the HCDR regions Determined according to Chothia or Kabat, or according to Chothia and Kabat (Chothia & Kabat).
  • the three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 of the present invention are selected from
  • the LCDR is determined according to Chothia or Kabat, or according to Chothia and Kabat (Chothia & Kabat).
  • the HCDR1 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO:3, 16, 28, 43, 55, 77, 90 or 119, or the HCDR1 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3, 16, 28, 43, 55, 77, 90 or 119.
  • the HCDR1 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:6, 19, 30, 46, 58, 80, 93, 122, or 107, or the HCDR1 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6, 19, 30, 46, 58, 80, 93, 122, or 107.
  • the HCDR2 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO:4, 17, 44, 56, 67, 78, 91, 120 or 85, or the HCDR2 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, 17, 44, 56, 67, 78, 91, 120 or 85.
  • the HCDR2 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:7, 20, 31, 37, 47, 59, 69, 81, 94, 103, 111, 123, 98 or 108, or the HCDR2 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7, 20, 31, 37, 47, 59, 69, 81, 94, 103, 111, 123, 98 or 108.
  • the HCDR3 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO: 5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86, or the HCDR3 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86.
  • the HCDR3 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86, or the HCDR3 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86.
  • LCDR1 determined based on Chothia and Kabat comprises an amino acid sequence of SEQ ID NO: 9, 21, 48, 60, 70, 82, 95, 104, 112 or 124, or consists of the amino acid sequence
  • LCDR1 determined based on Chothia and Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9, 21, 48, 60, 70, 82, 95, 104, 112 or 124.
  • LCDR2 determined based on Chothia and Kabat comprises an amino acid sequence of SEQ ID NO: 10, 22, 49, 61, 71, 83, 96, 105, 113, 125 or 52, or consists of the amino acid sequence
  • LCDR2 determined based on Chothia and Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, 22, 49, 61, 71, 83, 96, 105, 113, 125 or 52.
  • the LCDR3 determined based on Chothia and Kabat comprises or consists of the amino acid sequence of SEQ ID NO: 11, 23, 32, 38, 50, 62, 72, 84, 97, 106, 114 or 12, or the LCDR3 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11, 23, 32, 38, 50, 62, 72, 84, 97, 106, 114 or 12.
  • the antibody heavy chain constant region of the present invention is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1.
  • the antibody light chain constant region of the present invention is a lambda or kappa light chain constant region, preferably a kappa light chain constant region.
  • the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment in the form of IgG1 and comprises a human kappa light chain constant region.
  • the heavy chain constant region of the antibody of the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:63; or
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 63; or
  • (b) comprises or consists of an amino acid sequence selected from SEQ ID NO:73; or
  • (c) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 73.
  • amino acid changes occur in the Fc region.
  • the Fc region of the present invention is the Fc region of the present invention.
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 8;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 8; or
  • (b) comprises or consists of an amino acid sequence selected from SEQ ID NO:87; or
  • (c) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:87.
  • the antibody light chain constant region of the invention is provided.
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:64; or
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:64.
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises:
  • said HCDR and said LCDR are determined according to Chothia or Kabat, or according to Chothia and Kabat;
  • the HCDR is determined according to the Kabat scheme
  • the LCDR is determined according to the Kabat and Chothia combined scheme (Chothia &Kabat);
  • the HCDR is determined according to the Chothia scheme
  • the LCDR is determined according to the combined Kabat and Chothia scheme (Chothia & Kabat).
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises: complementary determining regions HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 are determined based on the Chothia scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs, and the LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combined scheme (Chothia & Kabat), and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs:
  • HCDR1, HCDR2 and HCDR3 are determined based on the Kabat scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs
  • LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combined scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs:
  • the anti-FR ⁇ antibody or antigen-binding fragment thereof of the present invention comprises VH and VL, wherein VH and VL respectively comprise the amino acid sequence shown in SEQ ID NO as listed in the following table, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO as listed in the following table, or consist of the amino acid sequence:
  • the present invention provides an anti-FR ⁇ antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprised by the antibody or the antigen-binding fragment thereof comprise or consist of the amino acid sequence shown in the following table SEQ ID NO:
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described in the present invention occur in regions outside the CDR (eg, in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the substitution is a conservative substitution.
  • a conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.
  • the substitution occurs in the CDR region of the antibody.
  • the variant obtained has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and/or will have certain biological properties that are substantially retained by the parent antibody.
  • the potential risk of molecular isomerization of the antibody is eliminated by substitution at the isomerization site, for example, a CDR, such as a light chain CDR, such as a D mutation in a light chain CDR2, for example, can be mutated to E, for example, the D at position 56 is mutated to E; for example, in the Pha3-HZ2-D/EG-VK of the antibody of the present invention, the D in the CDR2 of the light chain is mutated to E, D56E (Kabat).
  • cysteine engineered antibodies eg, "thioMAbs”
  • cysteine residues of an antibody are replaced with cysteine residues.
  • the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available.
  • Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl Cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (e.g. glycerol), polyvinyl alcohol,
  • PEG poly
  • the antibodies of the present invention are humanized. Humanization can be achieved by replacing one or more amino acid residues in the heavy chain variable region and light chain variable region of a natural antibody of non-human origin, especially the framework region sequence, with residues at corresponding positions in the variable region of a conventional antibody from a person. Methods for humanizing antibodies are well known in the art. Typically, humanization substitutions are carried out in a manner that maintains the favorable binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, etc., are well known in the art to determine and select suitable humanized residue mutations or mutation combinations.
  • the humanized antibody of the present invention can be obtained by a method comprising the following steps:
  • the human germline gene is the IGVH/IGKV germline gene; constructing the CDR region of the chimeric antibody onto the human framework region;
  • the light chain variable region may contain Q79E and/or Y87F and/or F71Y
  • the heavy chain variable region may contain A24G, such as Q79E and Y87F (Kabat numbering) in the light chain variable region of 2F22-HZ1, A24G (Kabat numbering) in the heavy chain variable region of Phage3-HZ1, and F71Y (Kabat numbering) in the light chain variable region;
  • Optimizing the amino acids at risk sites such as changing the isomerization sites to eliminate the isomerization risk, for example, the D in the CDR, such as the light chain CDR, such as the light chain CDR2 can be mutated, for example, to E, for example, the amino acid D in LCDR2 of the antibody Phage3-HZ2-D/EG-VK of the present invention is mutated to E, for example, wherein the amino acid D at position 56 is mutated to E (Kabat numbering); and
  • the anti-FR ⁇ antibodies or antigen-binding fragments thereof of the present invention further include antibodies or antigen-binding fragments having one or more of the following properties:
  • an "antibody that binds to the same or overlapping epitope as a reference antibody” refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks greater than 50%, 60%, 70%, 80%, 90% or 95% of the binding of that antibody to its antigen in a competition assay.
  • an antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay.
  • a reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay.
  • Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, biointerferometry (e.g., Fortebio), or surface plasmon resonance (Biacore), etc.
  • an antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more.
  • a reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more.
  • the binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that has at least 50%, 60%, 70%, 80%, 90% or 95% or more of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • the anti-FR ⁇ antibody of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.
  • the anti-FR ⁇ antibody is a monoclonal antibody.
  • the anti-FR ⁇ antibody is a chimeric antibody or a humanized antibody.
  • the anti-FR ⁇ antibodies of the present invention also encompass antibody fragments thereof, preferably selected from the following antibody fragments: antigen-binding fragments including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single domain antibody (sdAb), nanobody, sc(Fv)2.
  • antigen-binding fragments including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single domain antibody (sdAb), nanobody, sc(Fv)2.
  • the anti-FR ⁇ antibody molecule is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody can bind to two different epitopes of the FR ⁇ protein.
  • the bispecific antibody can bind to the FR ⁇ binding site and another protein.
  • the anti-FR ⁇ antibody is a bispecific antibody or a multispecific antibody.
  • the present invention provides nucleic acids encoding any of the above anti-FR ⁇ antibodies or fragments thereof or either chain thereof.
  • the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 14, 15, 26, 27, 35, 36, 41, 42, 53, 54, 65, 66, 75, 76, 88, 89, 100, 101, 109, 110, 117, 118, 13, 24, 25, 33, 34, 39, 40, 51, 74, 99, 115 or 116, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: NO:1, 2, 14, 15, 26, 27, 35, 36, 41, 42, 53, 54, 65, 66, 75, 76, 88, 89, 100, 101, 109, 110, 117, 118, 13, 24, 25, 33, 34, 39, 40, 51, 74, 99, 115 or 116 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of NO:
  • Each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences.
  • Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid phase DNA synthesis, or by PCR amplification.
  • the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any of their chains.
  • the polypeptide encoded by the nucleic acid can display human (and/or rhesus monkey) FR ⁇ antigen binding ability.
  • the nucleic acid encoding the variable region of the heavy chain and/or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and/or light chain, thereby generating nucleic acids encoding the antibody heavy chain and/or when expressed from a suitable expression vector.
  • a secretory signal peptide and/or a tag peptide that facilitates purification may be fused to the N-terminus of the heavy chain and/or light chain of the antibody.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • the vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).
  • the vector is a pCDNA vector, such as pCDNA3.1.
  • a host cell comprising the nucleic acid or the vector.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof.
  • the host cell is prokaryotic, e.g., bacteria, e.g., Escherichia coli.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies.
  • fungi and yeast strains that have been "humanized” in the glycosylation pathway lead to the production of antibodies with partial or complete human glycosylation patterns.
  • Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines modified to be suitable for suspension growth can be used.
  • useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO-S cells or CHO-K, etc.; and myeloma cell lines such as Y0, NS0 and Sp2/0.
  • Mammalian host cell lines suitable for producing antibodies are known in the art.
  • the present invention provides a method for preparing an anti-FR ⁇ antibody or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or a fragment thereof (preferably an antigen-binding fragment) or any one or both chains thereof, and optionally isolating the antibody or a fragment thereof (preferably an antigen-binding fragment).
  • the method further comprises recovering the anti-FR ⁇ antibody or a fragment thereof (preferably an antigen-binding fragment) from the host cell.
  • the polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and/or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
  • Antibodies prepared as described herein can be purified by known in the art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography such as Protein A, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the invention can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • anti-FR ⁇ antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as thin-layer interferometry, ELISA, etc. Binding to FR ⁇ can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or thin-layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used for measurement.
  • RIA radioimmunoassay
  • BBI thin-layer interferometry
  • ECL electrochemiluminescence
  • SPR surface plasmon resonance
  • FACS flow cytometry
  • the present invention also provides an assay for identifying anti-FR ⁇ antibodies with biological activity.
  • the biological activity is selected from the properties of the antibodies of the present invention, and may include, for example, binding to FR ⁇ (e.g., binding to human and/or rhesus monkey FR ⁇ ), endocytic activity, or killing activity of molecules comprising the antibody, etc.
  • the binding activity of the antibody molecules of the present invention to FR ⁇ or cells expressing FR ⁇ can be determined by methods known in the art, such as Fortebio, flow cytometry, Octet or Biacore, or the exemplary methods disclosed in the Examples herein.
  • the cellular (eg, FR ⁇ -positive cell) endocytosis activity of the antibody molecule of the present invention can be determined by methods known in the art, such as flow cytometry, or the exemplary methods disclosed in the Examples herein.
  • the killing activity of the antibody molecules of the present invention can be determined by methods known in the art, such as cell killing assays, such as in vitro cell killing assays, or the exemplary methods disclosed in the Examples herein.
  • the pharmacokinetic properties of the antibody molecules of the present invention can be determined by methods known in the art, such as the exemplary methods disclosed in the Examples herein.
  • Cells for use in any of the above in vitro assays include cell lines that naturally express FR ⁇ or are engineered to express FR ⁇ . Such cells also include cell lines that express FR ⁇ and are transfected with FR ⁇ -encoding DNA that does not normally express FR ⁇ .
  • any of the above assays can be performed using an anti-FR ⁇ antibody in combination with an additional active agent.
  • the present invention provides an immunoconjugate comprising any anti-FR ⁇ antibody provided herein and other substances, such as therapeutic agents or markers.
  • the therapeutic agent can be a therapeutic agent suitable for forming an immunoconjugate with FR ⁇ , such as an immunomodulator, such as an anti-inflammatory agent or an immunosuppressant.
  • the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a small molecule drug, an immunomodulator (e.g., an immunosuppressant) or other antibodies.
  • the immunoconjugate is used to prevent or treat FR ⁇ -related diseases, such as tumors or cancer.
  • the present invention provides a composition comprising any anti-FR ⁇ antibody or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof as described herein, preferably the composition is a pharmaceutical composition or a pharmaceutical preparation.
  • the composition further comprises a pharmaceutical excipient.
  • the composition e.g., a pharmaceutical composition, comprises an anti-FR ⁇ antibody or fragment thereof or an immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents (e.g., such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs or immunomodulators (e.g., immunosuppressants)).
  • the present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising a polynucleotide encoding an anti-FR ⁇ antibody.
  • the composition comprises one or more antibodies or fragments thereof that bind to FR ⁇ , or one or more polynucleotides encoding one or more anti-FR ⁇ antibodies or fragments thereof.
  • These compositions may also comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes and suppositories.
  • liposomes e.g., liposomes and suppositories.
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • compositions comprising the antibodies described herein can be prepared by mixing the antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.
  • the pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activity that will not adversely affect each other.
  • active ingredient e.g., chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants).
  • the active ingredient is suitably combined in an amount effective for the intended use.
  • the present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising an anti-FR ⁇ antibody or a fragment thereof (preferably an antigen-binding fragment) of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants).
  • therapeutic agents e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants).
  • Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, so that the dosage unit can be provided according to the dosage regimen or the interval of drug administration.
  • kit of parts of the present invention comprises in the same package:
  • a second container containing a pharmaceutical composition comprising another therapeutic agent, such as a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a vaccine, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).
  • another therapeutic agent such as a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a vaccine, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).
  • the combination product is used to prevent or treat FR ⁇ -related diseases, such as tumors or cancer.
  • the present invention provides a method for treating a FR ⁇ -related disease in a subject, comprising administering to the subject an effective amount of the anti-FR ⁇ antibody or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition or combination product of the present invention.
  • the FR ⁇ -related diseases described herein include tumors, such as cancer.
  • the cancer may be in the early, middle or late stages or may be metastatic cancer.
  • the cancer may be a solid tumor or a blood tumor.
  • the cancer is, for example, an epithelial cell cancer, such as lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumors, endometrial cancer, or cervical cancer.
  • the tumor refers to a tumor that highly expresses FR ⁇ in the tumor tissue or tumor cells of the individual.
  • the tumor refers to an increase in the protein level (e.g., expression) of FR ⁇ , or an increase in the nucleic acid level of FR ⁇ , in the tumor tissue or tumor cells of the individual, for example, compared with the adjacent normal tissue or normal cells of the individual (e.g., normal cells in the tissue) or the same tissue or cells therein of a healthy individual.
  • the present invention provides the use of an anti-FR ⁇ antibody or a fragment thereof in the production or preparation of a medicament for treating the relevant diseases or disorders mentioned herein.
  • the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of a disorder and/or symptoms associated with a disorder.
  • the preventive or therapeutic methods described herein further comprise administering to the subject or individual an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents.
  • treatment modalities include surgery, radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed to a preselected target or organ), or focused irradiation), etc.
  • radiation therapy e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed
  • local irradiation e.g., irradiation directed to a preselected target or organ
  • focused irradiation e.g., irradiation directed to a preselected target or organ
  • the therapeutic agent is selected from a chemotherapeutic agent, a cytokine, a cytotoxic agent, other antibodies, a vaccine, a small molecule drug, or an immunomodulator (eg, an immunosuppressant), among others.
  • the antibodies described herein can be combined with other antibodies for separate administration, e.g., separately as separate antibodies, or when linked (e.g., as a bispecific or multispecific antibody molecule).
  • Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or in separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agents and/or agents.
  • the anti-FR ⁇ antibodies of the present invention can be administered by any suitable method, including parenteral administration, and, if desired for local treatment, intralesional administration.
  • Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.
  • the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the anti-FR ⁇ antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, administration for preventive purposes, Whether administered for therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of any concomitant therapy and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • the present invention provides use of the anti-FR ⁇ antibody or fragment thereof, or an immunoconjugate or composition comprising the same, of the present invention in the production or preparation of a medicament for the uses described herein, such as for preventing or treating the relevant diseases or disorders mentioned herein.
  • the anti-FR ⁇ antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
  • therapies such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
  • the present invention also relates to methods for diagnosis and detection of the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosis and detection comprising the same.
  • any of the anti-FR ⁇ antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of FR ⁇ in a biological sample.
  • the term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR).
  • the biological sample is other liquid samples of blood, serum or biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from tumor tissue or cancer tissue.
  • an anti-FR ⁇ antibody for use in a method of diagnosis or detection is provided.
  • a method for detecting the presence of FR ⁇ in a biological sample comprises detecting the presence of FR ⁇ protein in a biological sample.
  • FR ⁇ is human FR ⁇ or rhesus monkey FR ⁇ .
  • the method comprises contacting the biological sample with an anti-FR ⁇ antibody as described herein under conditions that allow the anti-FR ⁇ antibody to bind to FR ⁇ , and detecting whether a complex is formed between the anti-FR ⁇ antibody and FR ⁇ . The formation of a complex indicates the presence of FR ⁇ .
  • the method can be an in vitro or in vivo method.
  • an anti-FR ⁇ antibody is used to select a subject suitable for treatment with an anti-FR ⁇ antibody, for example, wherein FR ⁇ is a biomarker for selecting the subject.
  • a method for detecting FR ⁇ in a sample comprising
  • Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detected moieties, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions.
  • directly detected labels or moieties such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels
  • indirectly detected moieties such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions.
  • the sample is obtained before treatment with an antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained before other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies.
  • FR ⁇ is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • a method for treating a disease of the present invention comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of FR ⁇ , thereby determining the FR ⁇ value, comparing the FR ⁇ value with a control value (e.g., a value in a healthy individual or normal tissue), and if the FR ⁇ value is greater than the control value, administering to the subject a therapeutically effective amount of an antibody or fragment thereof of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.
  • a subject e.g., a sample
  • a control value e.g., a value in a healthy individual or normal tissue
  • a method for treating a disease of the present invention comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of FR ⁇ , thereby determining the FR ⁇ value, comparing the FR ⁇ value with a control value (e.g., a value in a normal individual), and if the FR ⁇ value is greater than the control value, administering to the subject a therapeutically effective amount of the antibody or fragment thereof described in the present invention, or an immunoconjugate, composition, pharmaceutical composition, preparation, combination product, etc. comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.
  • a subject e.g., a sample
  • a control value e.g., a value in a normal individual
  • Myeloma cells P3X63Ag8.653 (Nanjing Kebai Company, catalog number CBP60876) were serially passaged using a culture medium containing 20 ug/mL 8-azaguanine (Merck #134-58-7) and cultured in a 5% CO 2 , 37° C. cell culture incubator.
  • mice were immunized using commercial recombinant human FR ⁇ -his protein (Nearshore Company catalog number C784) according to the conventional method described in the literature (Lonberg, N., et al., nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884).
  • Each mouse was immunized subcutaneously (SC) 4 times with recombinant human FR ⁇ .
  • Antigen-specific ELISA was used to determine the anti-FR ⁇ titer in the sera of immunized mice.
  • FR ⁇ solution with a concentration of 1ug/ml was coated in a 96-well plate, 100ul per well, and incubated overnight at 4°C. The coating solution was then washed off, and a blocking buffer (protein-free blocking buffer) was added to each well and incubated at room temperature for 2 hours.
  • Mouse serum was pre-diluted 100 times in PBSA (PBS solution containing 1% BSA) and diluted 10 times in a 1:2 dilution gradient. After washing off the coating solution, the diluted serum was added to the plate wells and incubated at room temperature for 1 hour.
  • PBSA PBS solution containing 1% BSA
  • mice with anti-FR ⁇ serum titers greater than 1:100,000 they were given an additional intraperitoneal (IP) booster immunization 4 days before fusion with 100 ⁇ g FR ⁇ solution dissolved in 100 ⁇ l PBS.
  • IP intraperitoneal
  • the constructed recombinant plasmids were transfected into Flp-In HEK293 cells (Invitrogen company #R758–07) and Flp-In CHO cells ( #R750–07 from Invitrogen), 48 hours after transfection, 100 ⁇ g/mL Hygromycin was used for screening and culture, 10 days later, the cells were sorted for monoclonal separation using a flow cytometer, and monoclonal clones with good growth were selected and identified using antibodies that specifically bind to FR ⁇ and expanded for culture, thus obtaining CHO cells overexpressing hFR ⁇ (named CHO-hFOLR1), HEK293 cells (named HEK293-hFOLR1 or HEK293-FR ⁇ ), and CHO cells overexpressing rhesus monkey FR ⁇ (named CHO-cynoFOLR1) for cytological experiments.
  • CHO-hFOLR1 CHO-hFOLR1
  • Example 2 Take the mouse spleen obtained in Example 1, and fuse the separated spleen cells with myeloma cells P3X63Ag8.653 using the electrofusion method.
  • the fused hybridoma cells were inoculated in a 384-well plate, cultured for 14 days, coated with human FR ⁇ -His recombinant protein (1ug/ml, pH9.6, 0.1M NaHCO3), incubated at 4°C overnight; blocked with 4% skim milk powder-PBS, incubated at 37°C for 2 hours; washed three times with PBST (0.05% Tween20-PBS), added with hybridoma clone culture supernatant, and incubated at 37°C for 1 hour.
  • human FR ⁇ -His recombinant protein (1ug/ml, pH9.6, 0.1M NaHCO3
  • PBST 0.05% Tween20-PBS
  • CHO-hFOLR1 The flow cytometry binding ability of CHO cells (CHO-hFOLR1) overexpressing hFR ⁇ was detected using a 96-well plate. 2 ⁇ 10 5 cells were used in each test well, and 20ul of hybridoma supernatant was added. After incubation at 4°C for 1 hour, the cells were washed with PBS; a 1000-fold diluted FITC-labeled anti-human antibody secondary antibody (Jackson #109-095-098) was added, and the cells were resuspended at 100mL/well. After incubation at 4°C for 1 hour, the cells were washed with PBS and analyzed on a flow cytometer (Figure 3).
  • CHO cells (CHO-cynoFOLR1) overexpressing cyno FR ⁇ were used to perform flow cytometry screening on the supernatant of candidate clones. After detection and identification, a total of multiple hybridoma clones that secreted positive FR ⁇ antibodies and recognized monkey FR ⁇ molecules were obtained. ( Figure 4)
  • hybridoma cell clones that specifically recognize human FR ⁇ obtained by binding experiment screening were subcloned into single cells by limiting dilution method. After two rounds of subcloning, each hybridoma cell clone obtained secreted only one antibody.
  • RNAfast200 kit Shanghai Feijie Biotechnology Co., Ltd.
  • 5 ⁇ PrimeScript RT Master Mix Takara was used to reverse transcribe the total RNA of the hybridoma cells into cDNA
  • degenerate primers were used (Anke Krebber.1997) and Extaq PCR reagent (Takara) were used to amplify the antibody light chain variable region VL and heavy chain variable region VH sequences
  • the PCR amplification products were purified using a PCR clean-up Gel extraction kit (Macherey-Nagel); according to the instructions of the pClone007 Simple Vector Kit (Qingke Biotechnology Co., Ltd.), the amplified PCR products were connected to the T vector and transformed into Escherichia coli competent cells, and the monoclonal
  • the heavy chain variable region encoding nucleic acid sequence of the mouse anti-human FR ⁇ monoclonal antibody and the publicly published heavy chain constant region encoding nucleic acid sequence of the human monoclonal antibody IgG1 subclass (SEQ ID NO: 73) were spliced together and constructed into the mammalian cell expression vector pcDNA TM 3.1 (+) (Invitrogen # V790-20); the light chain variable region encoding nucleic acid sequence of the mouse anti-human FR ⁇ monoclonal antibody and the publicly published light chain constant region encoding nucleic acid sequence of the human monoclonal antibody ⁇ subclass (SEQ ID NO: 87) were spliced together and constructed into the mammalian cell expression vector pcDNA TM 3.1 (+) (Invitrogen # V790-20).
  • the constructed heavy chain vector and light chain vector of the anti-human ⁇ chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Protein A to obtain the anti-human FR ⁇ chimeric antibody protein.
  • PEI polyethyleneimine
  • a similarly constructed control antibody MIRV (US20200362029A1) was constructed.
  • the SEQ ID NO of the variable region sequence and CDR sequence of the obtained chimeric antibody is shown in Table 1.
  • the antibody number or the antibody number -xiIgG will be used, which both represent chimeric antibodies having the variable region under the antibody number shown in Table 1 and an IgG1 heavy chain constant region and a ⁇ subclass light chain constant region.
  • the binding kinetic parameters of chimeric antibodies and antigen human FR ⁇ were analyzed using the Fortebio (BLITZ pro1.1.0.28) instrument.
  • the AHC biological probe (AHC, Satorius #18-5064) was soaked in PBS for 10 minutes; then the probe was placed in 100nM antibody, with a solidification height of 1nm, and the probe was further combined with 100nM antigen for 400 seconds; then the probe was transferred to PBS for a dissociation reaction for 600 seconds.
  • the binding of candidate antibodies was evaluated using human non-small cell lung cancer cells NCI-H2110 (Nanjing Kebai #CBP60071) and human ovarian cancer cells SKOV3 (Nanjing Kebai #CBP60291) expressing FR ⁇ on the cell membrane surface. 2 ⁇ 10 5 cells were used for each antibody sample to be tested, and the cells were washed twice with PBS.
  • the FR ⁇ test antibody was diluted with 1% BSA to three gradient points: high (0.4ug/mL), medium (0.1ug/mL), and low (0.01ug/mL), and the cells were resuspended with the diluted antibody; incubated at 4°C for 1 hour and washed with PBS; the FITC-labeled anti-human antibody secondary antibody (Jackson #109-095-098) was diluted 1000 times and used (10ul antibody solution, added with 9.99mL PBS to dilute to 10mL working solution), the cells were resuspended in 1000mL/well, and incubated at 4°C for 1 hour; after washing with PBS, the cells were analyzed by flow cytometer (iQue Plus, Satorius) ( Figures 10-13).
  • the antibodies of the present invention especially 2F22, Pha3, etc., bind to cells at different concentrations and show binding abilities comparable to those of the control antibody MIRV (Mirvecuximab).
  • the in vitro cellular endocytosis experiment of FR ⁇ chimeric antibody was evaluated using human non-small cell lung cancer cells NCI-H2110 as target cells.
  • the antibody to be tested was FITC fluorescently labeled using DOJINDO's LK01 kit.
  • the labeled antibody was diluted to 5 ⁇ g/mL using 1% BSA.
  • NCI-H2110 cells were cultured to the best state. 2 ⁇ 10 5 cells were used for each sample.
  • the cells were washed twice with PBS and resuspended in 2% BSA; the cells and the antibody to be tested were mixed at a volume of 1:1 and divided into three portions, two of which were placed on ice and one in a 37°C incubator for 4.5 hours.
  • endocytosis efficiency (37°C MFI - 4°C MFI) ⁇ 4°C MFI (PBS) ⁇ 100%
  • ADC was prepared as follows:
  • the humanized antibody heavy chain variable region sequence is similar to that of the humanized antibody.
  • the antibody framework region sequence is selected as a template, and the mouse antibody heavy chain CDR is combined with the human antibody framework region to finally generate the humanized antibody heavy chain variable region sequence.
  • the same process is used to generate the humanized antibody light chain variable region sequence, and the individual amino acids in the framework region are changed from human to mouse.
  • the engineered humanized antibody variable region heavy chain gene was constructed into a mammalian cell expression vector pcDNA TM 3.1(+)(invitrogen#V790-20) containing a heavy chain constant region gene of the human monoclonal antibody IgG1 subclass (SEQ ID NO: 73); the light chain variable region gene was constructed into a mammalian cell expression vector containing a light chain constant region gene of the human monoclonal antibody ⁇ subclass (SEQ ID NO: 87).
  • the constructed anti-human FR ⁇ humanized antibody heavy chain vector and light chain vector were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Protein A to obtain the anti-human FR ⁇ humanized antibody protein.
  • PEI polyethyleneimine
  • Table 8 VH, VL and CDR sequences of humanized antibodies.
  • the binding kinetic parameters of humanized antibody and antigen human FR ⁇ were analyzed using Fortebio (BLITZ pro1.1.0.28).
  • the AHC bioprobe was soaked in PBS for 10 minutes; The probe was placed in 100nM antibody, with a solidification height of 1nm, and the probe was further combined with 100nM antigen for 400 seconds; then the probe was transferred to PBS for dissociation for 600 seconds.
  • the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed using the software to calculate the kinetic constants of antigen-antibody binding ( Figures 22, 23; Tables 9-10).
  • the antibody-antigen interaction force was determined using the GE BIAcore instrument S200.
  • the anti-human Fc antibody GE Human Antibody Capture Kit. Cat#BR-1008-39
  • the sensor chip CM5 analysis channel and the control sample channel capture the human FR ⁇ antibody sample (100nM)
  • the gradient dilution of human FR ⁇ antigen Human FOLR1, near shore protein #C784
  • rhesus monkey FR ⁇ NCBI: NP_001181576.1
  • starting concentration 100nM, 1:2 gradient dilution was passed through the analysis channel and the control sample channel together to determine the light reaction value after the antibody-antigen binding.
  • the binding constant Kon and dissociation constant Koff of the antibody, as well as the affinity constant KD were finally obtained. The results are shown in Figures 24 and 25 and Tables 11-12.
  • the affinity of hz45A3 and hz45B1 molecules to human and monkey FRa is higher than that of the control molecule (MIRV, Mirvetuximab).
  • HEK293 cells overexpressing FR ⁇ , HEK293-hFOLR1, and oral epithelial cancer cells KB were used to evaluate the binding of humanized antibodies. 2 ⁇ 10 5 cells were used for each sample and the cells were washed twice with PBS. The FR ⁇ test antibody was diluted with 1% BSA to three gradient points: high (10 ⁇ g/mL), medium (1 ⁇ g/mL), and low (0.1 ⁇ g/mL).
  • the cells were resuspended with the diluted antibody; incubated at 4°C for 1 hour and washed with PBS; the FITC-labeled anti-human antibody secondary antibody (Jackson#109-095-098) was diluted 1000 times and the cells were resuspended in 100mL/well and incubated at 4°C for 1 hour; after washing with PBS, the cells were analyzed by flow cytometer ( Figures 26, 27).
  • the engineered molecules showed different affinities, among which molecules such as 2F22-H1L0 and Pha3-H1L2 showed FRa+ cell binding ability that was better than or equivalent to the benchmark molecules.
  • HEK293 cells overexpressing FR ⁇ , HEK293-hFOLR1 (HEK293-hFR ⁇ )_, and oral epithelial cancer cells KB were used to evaluate the binding of humanized antibodies. 2 ⁇ 10 5 cells were used for each sample and the cells were washed twice with PBS. The FR ⁇ test antibody was diluted 1:4 with FBS solution containing 1% BSA. 200ul of antibody solution, with a starting concentration of 10ug/mL, was added to the next dilution tube containing 400uL FBS_1% BSA solution in sequence, resulting in a total of 7 dilution concentrations.
  • the cells were resuspended to 100uL with the diluted antibody, incubated at 4°C for 1 hour, and then washed with PBS; the secondary antibody (Jackson#109-095-098) of FITC-labeled anti-human antibody diluted 1000 times with FBS_1% BSA was used, and the cells were resuspended at 100uL/well, incubated at 4°C for 1 hour, and then washed with PBS.
  • the analysis was performed using a flow cytometer ( FIGS. 28 and 29 and Tables 13 and 14 ).
  • hz45A3, hz45B1 and other molecules showed better binding to cells than control molecules (Mirv, Mirvetuximab) The ability of surface FRa.
  • Human non-small cell lung cancer cells NCI-H2110 were used as target cells to evaluate the in vitro cellular endocytosis ability of FR ⁇ humanized antibodies.
  • the antibody to be tested was FITC fluorescently labeled using DOJINDO's LK01 kit.
  • the labeled antibody was diluted to 5 ⁇ g/mL with 1% BSA.
  • 2E5 cells were used for each sample.
  • the cells were washed with PBS and resuspended in 2% BSA; the cells and the antibody to be tested were mixed at a volume of 1:1 and divided into three parts, two of which were placed on ice and one in a 37°C incubator for 4.5 hours.
  • endocytosis efficiency (37°C MFI - 4°C MFI) ⁇ 4°C MFI (PBS) ⁇ 100%
  • the engineered antibody molecules showed the ability to induce FRa+ cell endocytosis, among which 2F22-H1L0, 2F22-H1L1, Phage3-H1L2, Hz45A3 and Hz45B1 molecules all showed FRa+ cell endocytosis ability better than the control molecule (MIRV, Mirvetuximab).
  • Pha-H0L0-ADC (also expressed as hzPha-H0L0-MMAE), Pha-H0L1-ADC (also expressed as hzPha-H0L1-MMAE), Pha-H1L2-ADC (also expressed as hzPha-H1L2-MMAE), 2F22-H1L0-ADC (also expressed as 2F22-H1L0-MMAE), 2F22-H1L1-ADC (also expressed as 2F22-H1L1-MMAE), hz45A3-ADC (also expressed as hz45A3-MMAE), hz45B1-ADC (also expressed as hz45B1-MMAE), MIRV-ADC (also expressed as MIRV-MMAE or Mirve-MMAE) and NC-ADC (IgG1-MMAE) were prepared.
  • the absorbance of the conjugate at 248nm to 280nm was measured by ultraviolet spectrophotometer, and the concentration of the conjugate was calculated.
  • the samples were divided into cryovials and stored at -80°C.
  • the DAR values of the samples were determined by HPLC-HIC.
  • FR ⁇ humanized antibody ADC In vitro cell killing evaluation of FR ⁇ humanized antibody ADC was performed using HEK293-FR ⁇ cells and KB cells (ATCC#CCL-17). The density of HEK293-FR ⁇ cells and KB cells was adjusted to 5 ⁇ 10 4 /mL, and 100 ⁇ L/well was plated in a 96-well white plate. On the second day of the experiment, the antibody ADC molecule to be tested was diluted to 10 ⁇ g/mL using culture medium, and then Dilution was performed by 10 times, and 10 gradients were set. 50 ⁇ L/well was added to the cell culture plate and incubated in a carbon dioxide incubator for 96 hours.
  • the ADCs prepared by engineered molecules exhibited FRa+ cell killing ability, among which ADC molecules such as 2F22-H1L1, Pha3-H1L2, Hz45A3 and Hz45B1 showed FRa+ cell killing ability comparable to that of the control molecule (MIRV Mirvetuximab).
  • the antibodies to be tested (hzPha3H1L2, hz45B1, hz45A3, MIRV) were diluted to a concentration of 2 mg/mL with 0.9% sodium chloride injection. Five male SD rats were used for each antibody, and the required volume was calculated according to 10 mg/Kg.
  • hzPha3H1L2, hz45B1, and hz45A3 molecules have similar pharmacokinetic properties to the control molecule (MIRV, Mirvetuximab) in rats.
  • mice Four-week-old female BALB/c nu/nu nude mice were subcutaneously inoculated with KB cells. After the tumor grew to 100-150 mm3 , they were divided into 8 groups according to the tumor volume.
  • the test drugs (hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, Mirve-MMAE) were prepared with PBS at 0.1 mg/mL and 0.3 mg/mL, and intravenously (IV) injected with an injection volume of 0.1 mL/10 g body weight. The grouping and dosing are shown in Table 17.
  • the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume (V) was calculated as follows:
  • V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width respectively.
  • T/C (%) - (TT 0 ) / (CC 0 ) ⁇ 100, where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment, C represents the control group, and T represents the tumor volume of the experimental group.
  • T ⁇ T 0 or C ⁇ C 0 it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
  • the animals were killed by CO2 anesthesia, and then the tumors were dissected and photographed.
  • hzPha3-H1L2-MMAE (1, 3 mg/kg, IV, D0) significantly inhibited the growth of subcutaneous transplanted tumors in KB nude mice in a dose-dependent manner, with tumor inhibition rates of 64% and 98%, respectively.
  • 3 mg/kg dose group 5/6 mice had partial tumor regression; hz45B1-MMAE (1, 3 mg/kg, IV, D0) had tumor inhibition rates of 59% and 128%, respectively.

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Abstract

Provided are an antibody that specifically recognizes FRα or an antigen-binding fragment thereof, as well as a corresponding nucleic acid, a host cell, a drug, treatment and diagnosis methods, or a use.

Description

一种靶向FRα的抗体或其抗原结合片段及其应用An antibody or antigen-binding fragment targeting FRα and its application 技术领域Technical Field
本发明属于抗体领域，具体地涉及一种FRα结合分子，特别是特异性识别FRα的抗体和其片段。此外，本发明还涉及包含此类抗体或其片段的核酸或宿主细胞，包含此类抗体或其片段的药物，以及应用这些抗体和片段的治疗和诊断方法或用途。The present invention belongs to the field of antibodies, and specifically relates to a FRα binding molecule, in particular an antibody and a fragment thereof that specifically recognizes FRα. In addition, the present invention also relates to a nucleic acid or host cell containing such an antibody or a fragment thereof, a drug containing such an antibody or a fragment thereof, and a treatment and diagnosis method or use using these antibodies and fragments.
背景技术Background technique
叶酸受体α(Folate receptorα，FRα)是一种分子量大约40KD的细胞表面糖蛋白，最初是作为叶酸结合蛋白发现。FRα是叶酸受体(FRs)家族的一个成员，FRs家族还包括FRβ、FRγ和FRδ。叶酸-FRα复合物内吞入细胞进入内体小泡后，配体受体分离，FRα将再循环至细胞膜。膜型FRα会在酶切作用下脱落，形成可溶型FRα。FRα在实体瘤中有广泛高表达，比如间皮瘤(72-100％)，三阴性乳腺癌(35-68％)，卵巢癌(76-89％)，非小细胞肺癌(14-74％)；在非恶性组织中，支气管上皮细胞，脉络丛，甲状腺，唾液腺，乳腺，结肠，膀胱也有一定比例的FRα表达。叶酸受体参与肿瘤的浸润、转移、进展，成为肿瘤治疗有吸引力的靶点。Folate receptor α (FRα) is a cell surface glycoprotein with a molecular weight of about 40KD. It was originally discovered as a folate binding protein. FRα is a member of the folate receptor (FRs) family, which also includes FRβ, FRγ and FRδ. After the folate-FRα complex is internalized into the cell and enters the endosomal vesicle, the ligand receptor separates and FRα is recycled to the cell membrane. The membrane-type FRα will fall off under the action of enzyme cleavage to form a soluble FRα. FRα is widely and highly expressed in solid tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), and non-small cell lung cancer (14-74%); in non-malignant tissues, bronchial epithelial cells, choroid plexus, thyroid, salivary glands, breast, colon, and bladder also express a certain proportion of FRα. Folate receptors are involved in the infiltration, metastasis, and progression of tumors, making them attractive targets for tumor treatment.
已经有公司例如ImmunoGen通过基于杂交瘤平台，筛选得到针对人FRα的单克隆抗体Mirvetuximab，继而通过可切割连接子连接一个细胞毒性药物DM4，开发了Mirvetuximab soravtansine(IMGN853)。Morphotek(后被卫材收购)公司也开发针对人FRα的单克隆抗体Farletuzumab。Some companies, such as ImmunoGen, have screened the monoclonal antibody Mirvetuximab targeting human FRα based on a hybridoma platform, and then connected a cytotoxic drug DM4 through a cleavable linker to develop Mirvetuximab soravtansine (IMGN853). Morphotek (later acquired by Eisai) is also developing Farletuzumab, a monoclonal antibody targeting human FRα.
尽管已经开发了某些抗FRα抗体，但是对于在现有技术基础上进一步开发FRα抗体仍然存在需求，尤其是对于开发具有高特异性、高生物学活性、高内吞能力、更好的体内安全性，和/或可以用于制备ADC，CAR-T，CD3-engager的抗人FRα抗体，仍然存在需求。Although certain anti-FRα antibodies have been developed, there is still a need for further developing FRα antibodies based on the existing technology, especially for developing anti-human FRα antibodies with high specificity, high biological activity, high endocytosis ability, better in vivo safety, and/or that can be used to prepare ADC, CAR-T, CD3-engager.
发明内容Summary of the invention
本发明公开了一种靶向FRα的抗体或其抗原结合片段及其应用。The invention discloses an antibody or an antigen binding fragment thereof targeting FRα and application thereof.
本发明因此提供了一种新的结合FRα的抗体，以及其抗原结合片段。The present invention therefore provides a novel antibody that binds to FRα, and an antigen-binding fragment thereof.
在一些实施方案中，本发明的抗FRα抗体具有以下一个或多个或全部的特性：In some embodiments, the anti-FRα antibodies of the invention have one or more or all of the following properties:
(1)能够以高亲和力结合人或恒河猴FRα；(1) Ability to bind human or rhesus monkey FRα with high affinity;
(2)能够以高亲和力结合细胞膜表面表达的人或恒河猴FRα；(2) Ability to bind to human or rhesus monkey FRα expressed on the cell membrane surface with high affinity;
(3)能够诱导FRα阳性细胞对抗体或其片段或包含所述抗体或其片段的分子的内吞作用，优选地具有相当于已知抗FRα抗体(例如MIRV)或优于已知抗FRα抗体(例如MIRV)的内吞效率；(3) capable of inducing endocytosis of the antibody or fragment thereof or a molecule comprising the antibody or fragment thereof by FRα-positive cells, preferably with an endocytosis efficiency equivalent to or better than that of known anti-FRα antibodies (e.g., MIRV);
(4)包含其的分子具有细胞(例如肿瘤细胞，例如FRα阳性细胞，例如FRα阳性肿瘤细胞)杀伤能力。(4) The molecule comprising the same has the ability to kill cells (eg, tumor cells, eg, FRα-positive cells, eg, FRα-positive tumor cells).
在一些实施方案中，本发明提供了编码本发明抗体或其片段的核酸，包含所述核酸的载体，包含所述载体的宿主细胞。 In some embodiments, the present invention provides nucleic acids encoding the antibodies or fragments thereof of the present invention, vectors comprising the nucleic acids, and host cells comprising the vectors.
在一些实施方案中，本发明提供了制备本发明抗体或其片段的方法。In some embodiments, the invention provides methods of making an antibody or fragment thereof of the invention.
在一些实施方案中，本发明提供了包含本发明抗体的免疫缀合物、药物组合物和组合产品。In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions and combination products comprising the antibodies of the invention.
本发明还提供了利用本发明抗体在受试者中阻断FRα介导的信号通路的方法，以及预防或治疗FRα相关疾病，例如免疫系统疾病(例如自身免疫疾病或炎症)的方法。The present invention also provides methods for blocking FRα-mediated signaling pathways in subjects using the antibodies of the present invention, as well as methods for preventing or treating FRα-related diseases, such as immune system diseases (eg, autoimmune diseases or inflammation).
本发明还涉及在样品中检测FRα的方法。The present invention also relates to a method for detecting FRα in a sample.
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are easily conceivable to those skilled in the art will be included in the spirit of the present invention and the protection scope of the appended claims.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示了小鼠血清FRα抗体滴度(650nM处测量的吸光度)；Figure 1 shows the mouse serum FRα antibody titer (absorbance measured at 650 nM);
图2显示了FRα杂交瘤上清与人FRα-His结合的ELISA检测；Figure 2 shows the ELISA detection of FRα hybridoma supernatant binding to human FRα-His;
图3显示了杂交瘤上清与过表达hFRα的CHO细胞(CHO-hFOLR1)结合的FACS检测；FIG3 shows FACS detection of the binding of hybridoma supernatants to CHO cells overexpressing hFRα (CHO-hFOLR1);
图4显示了杂交瘤上清与过表达cyno FRα的CHO细胞(CHO-cynoFOLR1)结合的FACS检测；Figure 4 shows the FACS detection of the hybridoma supernatant binding to CHO cells overexpressing cyno FRα (CHO-cynoFOLR1);
图5-图9显示了不同抗FRα嵌合抗体体外结合人FRα的能力，其中抗体编号-xiIgG(Sample ID-xiIgG)是指如实施例2.6所制备的具有IgG1的重链恒定区和κ亚类轻链恒定区的嵌合抗体，其可变区参见表1中对应“抗体编号(即Sample_ID)”的可变区；MIRV：Mirvecuximab，其作为阳性对照(PC)；NC：阴性对照，IgG1(序列见序列表SEQ ID NO:63和SEQ ID NO:64)。Figures 5-9 show the ability of different anti-FRα chimeric antibodies to bind to human FRα in vitro, where Antibody No.-xiIgG (Sample ID-xiIgG) refers to the chimeric antibody having the heavy chain constant region of IgG1 and the light chain constant region of the κ subclass prepared as in Example 2.6, and its variable region refers to the variable region corresponding to "Antibody No. (i.e., Sample_ID)" in Table 1; MIRV: Mirvecuximab, which serves as a positive control (PC); NC: negative control, IgG1 (sequence see sequence listing SEQ ID NO:63 and SEQ ID NO:64).
图10-图13显示了不同抗FRα嵌合抗体与不同细胞的结合能力，其中抗体编号-xiIgG是指如实施例2.6所制备的具有IgG1的重链恒定区和κ亚类轻链恒定区的嵌合抗体，其可变区参见表1中对应“抗体编号”的可变区；MIRV：Mirvecuximab，其作为阳性对照(PC)；NC：阴性对照，IgG1。Figures 10-13 show the binding ability of different anti-FRα chimeric antibodies to different cells, wherein antibody number -xiIgG refers to a chimeric antibody having a heavy chain constant region of IgG1 and a κ subclass light chain constant region as prepared in Example 2.6, and its variable region refers to the variable region corresponding to the "antibody number" in Table 1; MIRV: Mirvecuximab, which serves as a positive control (PC); NC: negative control, IgG1.
图14-图21显示了不同的抗FRα嵌合抗体的ADC的体外细胞杀伤结果(RLU曲线和IC50值)，其中抗体编号-ADC是指如实施例2.6所制备的具有IgG1的重链恒定区和κ亚类轻链恒定区的嵌合抗体与vcMMAE的ADC，MIRV-ADC是指MIRV的ADC分子MIRV-vcMMAE,NC-ADC是指IgG1与vcMMAE的ADC。Figures 14-21 show the in vitro cell killing results (RLU curves and IC50 values) of ADCs of different anti-FRα chimeric antibodies, wherein Antibody No.-ADC refers to the ADC of a chimeric antibody having a heavy chain constant region of IgG1 and a κ subclass light chain constant region and vcMMAE as prepared in Example 2.6, MIRV-ADC refers to the ADC molecule MIRV-vcMMAE of MIRV, and NC-ADC refers to the ADC of IgG1 and vcMMAE.
图22-23显示了如实施例3.1所制备的人源化抗体hz2F22-H1L1、hz2F22-H1L0、hzPha3-H0L0、hzPha3-H1L2、hzPha3-H1L1、hzPha3-H0L1和hzPha3-H1L0以及嵌合抗体Pha3-xiIgG和2F22-xiIgG与人FRα的Octet评估的结合动力学结果。Figures 22-23 show the Octet-assessed binding kinetics results of the humanized antibodies hz2F22-H1L1, hz2F22-H1L0, hzPha3-H0L0, hzPha3-H1L2, hzPha3-H1L1, hzPha3-H0L1 and hzPha3-H1L0 prepared as in Example 3.1 and the chimeric antibodies Pha3-xiIgG and 2F22-xiIgG with human FRα.
图24-25显示了如实施例3.1所制备的人源化抗体hz45A3和hz45B1以及对照抗体MIRV与人FRα(hFOLR1)和恒河猴FRα(cynoFOLR1)的Biacore评估的结合动力学结果。Figures 24-25 show the results of Biacore-assessed binding kinetics of the humanized antibodies hz45A3 and hz45B1 prepared as in Example 3.1 and the control antibody MIRV to human FRα (hFOLR1) and rhesus monkey FRα (cynoFOLR1).
图26-27显示了如实施例3.1所制备的人源化抗体hz2F22-H1L1、hz2F22-H1L0、hzPha3-H0L0、hzPha3-H1L2、hzPha3-H1L1、hzPha3-H0L1和hzPha3-H1L0以及嵌合抗体 Pha-xiIgG和2F22-xiIgG与FRα阳性细胞的结合能力，其中NC：阴性对照，IgG1；MIRV为阳性对照Mirvetuximab。Figures 26-27 show the humanized antibodies hz2F22-H1L1, hz2F22-H1L0, hzPha3-H0L0, hzPha3-H1L2, hzPha3-H1L1, hzPha3-H0L1 and hzPha3-H1L0 and chimeric antibodies prepared as in Example 3.1 The binding ability of Pha-xiIgG and 2F22-xiIgG to FRα-positive cells, where NC: negative control, IgG1; MIRV: positive control Mirvetuximab.
图28和29显示了如实施例3.1所制备的人源化抗体hz45A3和hz45B1与FRα阳性细胞的结合能力，其中NC：阴性对照，IgG1；MIRV为阳性对照Mirvetuximab。Figures 28 and 29 show the binding ability of humanized antibodies hz45A3 and hz45B1 prepared as in Example 3.1 to FRα-positive cells, wherein NC: negative control, IgG1; MIRV: positive control Mirvetuximab.
图30-35显示了不同的人源化抗体hz2F22-H1L1、hz2F22-H1L0、hzPha3-H0L0、hzPha3-H1L2、hzPha3-H1L1、hzPha3-H0L1和hzPha3-H1L0以及hz45A3和hz45B1的ADC的体外细胞(图30-34：HEK293-FRα(HEK293-hFOLR1)细胞；图35：KB细胞)杀伤结果(RLU曲线和IC50值)，其中抗体编号-ADC是指如实施例3.1所制备的具有IgG1的重链恒定区和κ亚类轻链恒定区的人源化抗体与vcMMAE的ADC，MIRV-ADC是指MIRV与vcMMAE的ADC分子。Figures 30-35 show the in vitro cell (Figures 30-34: HEK293-FRα (HEK293-hFOLR1) cells; Figure 35: KB cells) killing results (RLU curves and IC50 values) of different humanized antibodies hz2F22-H1L1, hz2F22-H1L0, hzPha3-H0L0, hzPha3-H1L2, hzPha3-H1L1, hzPha3-H0L1 and hzPha3-H1L0, as well as ADCs of hz45A3 and hz45B1, wherein Antibody No. -ADC refers to the ADC of a humanized antibody having a heavy chain constant region of IgG1 and a kappa subclass light chain constant region and vcMMAE as prepared in Example 3.1, and MIRV-ADC refers to the ADC molecule of MIRV and vcMMAE.
图36显示了不同抗体分子hz45A3和hz45B1和hzPha3-H1L2在体内的药代动力学，即随时间的浓度。FIG. 36 shows the pharmacokinetics of different antibody molecules hz45A3, hz45B1 and hzPha3-H1L2 in vivo, ie, the concentrations over time.
图37显示了hzPha3-H1L2-MMAE、hz45B1-MMAE、hz45A3-MMAE、Mirve-MMAE1mg/Kg给药组对人宫颈癌KB皮下移植瘤生长的影响。Figure 37 shows the effects of hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, and Mirve-MMAE1 mg/Kg administration groups on the growth of human cervical cancer KB subcutaneous transplanted tumors.
图38显示了hzPha3-H1L2-MMAE、hz45B1-MMAE、hz45A3-MMAE、Mirve-MMAE3mg/Kg给药组对人宫颈癌KB皮下移植瘤生长的影响。Figure 38 shows the effects of hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, and Mirve-MMAE 3 mg/Kg administration groups on the growth of human cervical cancer KB subcutaneous transplanted tumors.
发明详述DETAILED DESCRIPTION OF THE INVENTION
I.定义I. Definitions
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.
如本文所用，“FRα”、“叶酸受体α(FRα)”或“FOLR1”是指任何天然FRα多肽(例如人FRα多肽)或其变体。术语“FRα”涵盖“全长”未处理FRα多肽以及由细胞内的处理产生的任何形式的FRα多肽。所述术语还涵盖FRα的天然存在的变体，例如由剪切变体和等位基因变体编码的那些。本文所述的FRα多肽可从多种来源，诸如从人组织或从另一来源例如恒河猴分离，或通过重组或合成方法制备。在本发明的一个实施方案中，人FRα蛋白的-核苷 酸序列在中如登录号NM_000802.3所示。在本发明的一个实施方案中，恒河猴FRα蛋白的核苷酸序列在中如登录号NM_001194647.3所示。As used herein, "FRα", "folate receptor α (FRα)" or "FOLR1" refers to any natural FRα polypeptide (e.g., human FRα polypeptide) or variant thereof. The term "FRα" encompasses "full-length" unprocessed FRα polypeptides as well as any form of FRα polypeptides produced by processing within a cell. The term also encompasses naturally occurring variants of FRα, such as those encoded by splice variants and allelic variants. The FRα polypeptides described herein can be isolated from a variety of sources, such as from human tissue or from another source, such as rhesus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the -nucleoside of the human FRα protein The acid sequence is In one embodiment of the present invention, the nucleotide sequence of the rhesus monkey FRα protein is As shown in accession number NM_001194647.3.
本文所用的术语“抗FRα抗体”、“抗FRα”、“FRα抗体”或“结合FRα的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合(灵长类动物，例如人或恒河猴)FRα或其片段。As used herein, the terms "anti-FRα antibody," "anti-FRα," "FRα antibody," or "FRα-binding antibody" refer to an antibody that is capable of binding to (primate, such as human or rhesus monkey) FRα or a fragment thereof with sufficient affinity.
术语“全抗体”或“全长抗体”在本文中可互换使用，是指具有天然免疫球蛋白分子结构的抗体分子。在常规四链IgG抗体的情况下，全长抗体包含由二硫键相互连接的两条重链(H)和两条轻链(L)。在仅具有重链而缺乏轻链的重链抗体情况下，全长抗体包含由二硫键相互连接的两条重链(H)。对于常规四链IgG抗体，全长抗体重链通常由重链可变区(本文中缩写为VH)和重链恒定区组成，其中重链恒定区至少包含3个结构域CH1、CH2和CH3。全长抗体轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成，其中轻链恒定区由一个结构域CL组成。每个重链可变区VH和每个轻链可变区都由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。术语“抗体片段”包括完整抗体的一部分。在优选的实施方案中，抗体片段为抗原结合片段。The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies with only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.
术语抗体的“抗原结合片段”是与全长抗体不同的分子，其包含全长抗体的一部分，但其能结合全长抗体的抗原或与全长抗体(即与抗原结合片段所来源的全长抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、双体抗体(diabody)、单域抗体(sdAb)、纳米抗体、sc(Fv)2。例如，通过木瓜蛋白酶消化全长抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab')2，其为Fab’的二聚体，是二价的抗体片段。F(ab')2可以在中性条件下通过破坏铰链区中的二硫键而被还原，由此将F(ab')2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段。Fv片段由抗体单臂的VL和VH结构域组成。Fv片段的两个结构域VL和VH可以由独立的基因编码，但是也可以使用重组方法，使用合成性连接肽连接这两个结构域以使其作为单条蛋白链产生，并且在所述单条蛋白链中VL区和VH区配对以形成单链Fv(scFv)。sc(Fv)2是小型抗体，其中两个VH和两个VL通过接头连接形成单链。The term "antigen-binding fragment" of an antibody is a molecule different from a full-length antibody, which contains a portion of a full-length antibody, but which can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody, sc(Fv) 2 . For example, Fab fragments can be obtained by digesting full-length antibodies with papain. In addition, digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and is a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but recombinant methods can also be used to connect the two domains using synthetic connecting peptides to produce them as a single protein chain, and in the single protein chain, the VL region and the VH region are paired to form a single-chain Fv (scFv). sc(Fv)2 is a small antibody in which two VH and two VL are connected by a linker to form a single chain.
“Fab片段”或“Fab”在本文中可互换使用，用于指，由两条多肽链组成的、包含免疫球蛋白重链可变结构域VH、重链恒定结构域CH1、轻链可变结构域VL和轻链恒定结构域CL的免疫球蛋白片段，其中，一条多肽链从N端到C端包含VH和选自CH1和CL的一个恒定区，另一条多肽链从N端到C端包含VL和选自CL和CH1的另一恒定区，其中所述VH结构域和VL结构域配对形成抗原结合位点。在本文中，包含重链恒定区CH1的Fab多肽链也称作“Fab重链”；相应地，包含轻链恒定区CL的Fab多肽链也称作“Fab轻链”。"Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from N-terminus to C-terminus, and the other polypeptide chain comprises VL and another constant region selected from CL and CH1 from N-terminus to C-terminus, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as a "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as a "Fab light chain".
本文中的术语“Fc区”或“Fc结构域”用于定义含有至少一部分恒定区的免疫球蛋白重链的C端区域。所述术语包括天然序列Fc区和变体Fc区。在一个实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。然而，Fc区的C末端赖氨酸(Lys447)或甘氨酸-赖氨酸(残基446-447)可能存在，也可能不存在。除非本文另有说明，Fc区或恒定区中氨基酸残基的编号是根据EU编号系统，也称为EU索引，如Kabat等人,Sequences of Proteinsof Immunological Interest,第 5版Public Health Service,National Institutes ofHealth,Bethesda,MD,1991中所述。The term "Fc region" or "Fc domain" herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
“双体抗体”是通过基因融合构建的二价小型抗体，例如其是由两个多肽链组成的二聚体。双体抗体的每一多肽链的VL和VH域通过接头结合，从而编码在同一多肽链中的VL和VH形成具有不同的单链可变区片段的二聚体。双体抗体一般具有两个抗原结合位点。 "Diabodies" are small bivalent antibodies constructed by gene fusion, for example, dimers consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are bound by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region fragments. Diabodies generally have two antigen binding sites.
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派方案的任一种或其组合确定，所述指派方案包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (Kabat et al., Sequen ces of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures.
以下为采用kabat、AbM、Chothia、Contact和IMGT方案定义的CDR的区域范围(http://www.bioinf.org.uk/abs/info.html)。

The following are the regions of CDR defined using the Kabat, AbM, Chothia, Contact and IMGT schemes (http://www.bioinf.org.uk/abs/info.html).

除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
在一个实施方案中，本发明中抗体重链可变区的CDR是按照Chothia确定的。在一些实施方案中，本发明中抗体的重链可变区的CDR是按照Kabat确定的。在一些实施方案中，本发明中抗体的轻链可变区的CDR是按照Chothia和Kabat组合取最长后确定的(后文中显示为“Chothia&Kabat”)。In one embodiment, the CDR of the heavy chain variable region of the antibody of the present invention is determined according to Chothia. In some embodiments, the CDR of the heavy chain variable region of the antibody of the present invention is determined according to Kabat. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the longest combination of Chothia and Kabat (hereinafter shown as "Chothia & Kabat").
应该注意，基于不同的指派方案获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派方案下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派方案规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment schemes are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment scheme rules or combinations).
术语“嵌合抗体”是这样的抗体分子，其中(a)将恒定区或其部分改变、替换或交换，从而抗原结合位点与不同的或改变的类别、效应子功能和/或物种的恒定区或赋予嵌合抗体新性能的完全不同的分子(例如，酶、毒素、激素、生长因子、药物)等连接；或(b)将可变区或其部分用具有不同或改变的抗原特异性的可变区改变、替换或交换。例如，小鼠抗体可以通过将其恒定区更换为来自人免疫球蛋白的恒定区进行修饰。由于更换为人类恒定区，该嵌合抗体可以保留其在识别抗原方面的特异性，同时如与原始小鼠抗体相比，具有在人类中降低的免疫原性。The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and/or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.
“人源化抗体”是一种保留非人类抗体(例如小鼠单克隆抗体)的抗原特异性反应性，同时作为治疗药对人施用时免疫原性较低的抗体。这可以例如通过保留非人类抗原结合位点并将抗体的剩余部分替换成它们的人类相应部分(即，将恒定区以及可变区中不参与结合的部分替换为人类抗体的相应部分)来实现。A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the rest of the antibody with their human counterparts (i.e., replacing the constant region and the portion of the variable region that is not involved in binding with the corresponding portion of a human antibody).
如本文所用，术语“抗”、“结合”或“特异性结合”意指结合作用对靶标或抗原是选择性的并且可以与不想要的或非特异的相互作用区别。结合位点与特定靶标或抗原结合的能力可以通过流式细胞术或酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).
“免疫缀合物”是与一个或多个其他物质(例如大分子物质、放射性元素、细胞毒性剂等)缀合的抗体。An "immunoconjugate" is an antibody conjugated to one or more other substances (eg, macromolecular substances, radioactive elements, cytotoxic agents, etc.).
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。 "Individuals" or "subjects" include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses administered to a patient, produces the desired effect in a patient in need of treatment or prevention.
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数或改善可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%, relative to an untreated subject.
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity screened or selected in the initially transformed cells. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, and also include cells inside transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
“分离的”抗体或分子是这样的抗体或分子，其已经与其天然环境的组分分离。在一些实施方案中，将抗体或分子纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC).
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。在一些实施方案中，本发明考虑本发明抗体分子的变体，所述变体相对于在本文中具体公开的抗体分子及其序列而言具有相当程度的同一性，例如同一性为至少80％,85％,90％,95％,97％,98％或99％或更高。所述变体可以包含保守性改变。The "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, which have a considerable degree of identity, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes.
对于多肽序列，“保守性改变”包括对多肽序列的置换、缺失或添加，但不实质性改变多肽序列的期望功能活性。例如，保守性置换常常导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。以下列出了8组含有互为保守替换的氨基酸：1)丙氨酸(A)、甘氨酸(G)；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)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)。在一些实施方案中，术语“保守序列改变”用于指不显著影响或改变含有氨基酸序列的本发明抗体分子或结合蛋白分子的目的抗原结合特征的氨基酸修饰。例如保守修改变体相对于亲本抗体或结合蛋白保持对目的抗原至少80％,85％,90％,95％,98％,99％或更高，例如100-110％或更高的结合亲和力。 For polypeptide sequences, "conservative changes" include replacement, deletion or addition of polypeptide sequences, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.
“化疗剂”包括在治疗癌症或免疫系统疾病中有用的化学化合物。"Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。The term "small molecule drug" refers to low molecular weight organic compounds that can regulate biological processes. "Small molecules" are defined as molecules with a molecular weight less than 10kD, usually less than 2kD, and preferably less than 10kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包含免疫抑制剂。在一些实施方案中，本发明的免疫调节剂包括免疫检查点抑制剂或免疫检查点激动剂。The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.
术语“药物组合物”指这样的组合物，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，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
术语“药物组合或组合产品”是指非固定组合产品或固定组合产品，包括但不限于药盒/试剂盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的抗体、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit/test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and/or time interval of the two or more active agents are selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in a separate formulation, which can be the same or different.
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。The term "combination therapy" refers to the administration of two or more therapeutic agents or treatments to treat diseases described herein. This administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes the co-administration of each active ingredient in a variety of or in separate containers (e.g., tablets, capsules, powders, and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转疾病的症状、并发症、或生化指征的发作、缓解症状或阻止或抑制疾病、病状或病症的进一步发展。As used herein, "treat," ...
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。As used herein, "prevention" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。术语“表达载体”是指包含重组多核苷酸的载体，其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些，包括被掺入重组多核苷 酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including those incorporated into recombinant polynucleotides. The invention relates to cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) containing nucleic acids.
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如泪液、玻璃体液、脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。"Subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference.Any or all features discussed above and throughout this application can be combined in various embodiments of the present invention.In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive.Other features, purposes and advantages of the present invention will be apparent from this specification and accompanying drawings and from the appended claims.
II.抗体II. Antibodies
在一些实施方案中，本发明的抗FRα抗体或其片段以高亲和力结合灵长类动物FRα(例如人FRα或恒河猴FRα)，例如，以以下平衡解离常数(KD)与FRα(例如人FRα)结合，所述KD小于约40nM、35nM、30nM、25nM、20nM、15nM、10nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM或0.08nM，或在大约0.01nM或0.05nM至以上任一数值的范围内，或以上任一数值的范围内。In some embodiments, an anti-FRα antibody or fragment thereof of the invention binds primate FRα (e.g., human FRα or rhesus monkey FRα) with high affinity, e.g., binds to FRα (e.g., human FRα) with an equilibrium dissociation constant ( KD ) of less than about 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM , 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, or 0.08 nM, or within a range of about 0.01 nM or 0.05 nM to any of the above values, or within a range of any of the above values.
在一些实施方案中，本发明的抗体或其片段能够诱导FR诱阳性细胞对抗体或其片段或包含所述抗体或其片段的分子的内吞作用，优选地具有相当于已知抗FR细抗体(例如MIRV)或优于已知抗FR优抗体(例如MIRV)的内吞效率。In some embodiments, the antibodies or fragments thereof of the present invention are capable of inducing endocytosis of the antibodies or fragments thereof or molecules comprising the antibodies or fragments thereof by FR-positive cells, preferably with an endocytosis efficiency equivalent to or better than known anti-FR antibodies (e.g., MIRV).
在一些实施方案中，包含本发明的抗体或其片段的分子具有细胞(例如肿瘤细胞，例如FR些阳性细胞，例如FR细阳性肿瘤细胞)杀伤能力。In some embodiments, molecules comprising antibodies of the present invention or fragments thereof have cell (eg, tumor cell, eg, FR cell-positive cell, eg, FR cell-positive tumor cell) killing ability.
在一些实施方案中，本发明的抗体或其片段(任选地与治疗方式和/或其它治疗剂，例如免疫调节剂组合)能够预防或治疗FRα相关疾病，例如肿瘤，例如实体肿瘤，例如上皮细胞癌，例如肺癌(例如非小细胞肺癌)、口腔癌(如口腔上皮癌)、卵巢癌、乳腺癌、间质瘤、子宫内膜癌。In some embodiments, the antibodies or fragments thereof of the present invention (optionally in combination with treatment modalities and/or other therapeutic agents, such as immunomodulators) are capable of preventing or treating FRα-related diseases, such as tumors, such as solid tumors, such as epithelial cell cancers, such as lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumors, and endometrial cancer.
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region.
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。In some embodiments, an anti-FRα antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.
在一些方面中，本发明的抗FRα抗体或其抗原结合片段包含重链可变区(VH)。在一些方面中，本发明的抗FRα抗体或其抗原结合片段包含轻链可变区(VL)。在一些方面中，本发明的抗FRα抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3，例如通过Kabat和/或Chothia方案确定的HCDR1、HCDR2和HCDR3。 在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3，例如通过Kabat和/或Chothia方案(例如Chothia&Kabat方案)确定的HCDR1、HCDR2和HCDR3。In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, such as HCDR1, HCDR2, and HCDR3 determined by the Kabat and/or Chothia schemes. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3, such as HCDR1, HCDR2 and HCDR3 determined by the Kabat and/or Chothia scheme (e.g., the Chothia & Kabat scheme).
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段还包含抗体重链恒定区。在一些实施方案中，本发明抗FRα抗体或其抗原结合片段还包含抗体轻链恒定区。在一些实施方案中，本发明抗FRα抗体或其抗原结合片段还包含重链恒定区和轻链恒定区。In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region and a light chain constant region.
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段包含抗体重链(HC)。在一些实施方案中，本发明抗FRα抗体或其抗原结合片段还包含抗体轻链(LC)。在一些实施方案中，本发明抗FRα抗体或其抗原结合片段包含重链和轻链。In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises an antibody heavy chain (HC). In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain (LC). In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain.
在一些实施方案中，本发明的抗体重链包含抗体重链可变区和抗体重链恒定区。在一些实施方案中，本发明的抗体轻链包含抗体轻链可变区和抗体轻链恒定区。In some embodiments, the antibody heavy chain of the present invention comprises an antibody heavy chain variable region and an antibody heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises an antibody light chain variable region and an antibody light chain constant region.
在一些实施方案中，本发明的重链可变区In some embodiments, the heavy chain variable region of the invention
(i)包含与选自SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者(i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116; or
(ii)包含选自SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116; or
(iii)包含与选自SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116, and preferably, the amino acid changes do not occur in the CDR regions.
在一些实施方案中，本发明的轻链可变区In some embodiments, the light chain variable region of the invention
(i)包含与选自SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者(i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51; or
(ii)包含选自SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51; or
(iii)包含与选自SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51, and preferably, the amino acid changes do not occur in the CDR regions.
在一些实施方案中，本发明的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3选自In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the present invention, HCDR1, HCDR2 and HCDR3 are selected from
(i)如SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或(i) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116, or
(iv)相对于(i)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，优选地，所述HCDR根 据Chothia或Kabat确定，或根据Chothia和Kabat(Chothia&Kabat)确定。(iv) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to the sequence of any one of (i). Preferably, the HCDR regions Determined according to Chothia or Kabat, or according to Chothia and Kabat (Chothia & Kabat).
在一些实施方案中，本发明的3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3选自In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 of the present invention are selected from
(i)如SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，(i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51,
或or
(iii)相对于(i)中任一项的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列优选地，所述LCDR根据Chothia或Kabat确定，或根据Chothia和Kabat(Chothia&Kabat)确定。(iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i). Preferably, the LCDR is determined according to Chothia or Kabat, or according to Chothia and Kabat (Chothia & Kabat).
在一些实施方案中，基于Chothia确定的HCDR1包含SEQ ID NO:3、16、28、43、55、77、90或119的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia确定的HCDR1包含与SEQ ID NO:3、16、28、43、55、77、90或119的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR1 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO:3, 16, 28, 43, 55, 77, 90 or 119, or the HCDR1 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3, 16, 28, 43, 55, 77, 90 or 119.
在一些实施方案中，基于Kabat确定的HCDR1包含SEQ ID NO:6、19、30、46、58、80、93、122、或107的氨基酸序列，或由所述氨基酸序列组成，或者基于Kabat确定的HCDR1包含与SEQ ID NO:6、19、30、46、58、80、93、122、或107的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR1 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:6, 19, 30, 46, 58, 80, 93, 122, or 107, or the HCDR1 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6, 19, 30, 46, 58, 80, 93, 122, or 107.
在一些实施方案中，基于Chothia确定的HCDR2包含SEQ ID NO:4、17、44、56、67、78、91、120或85的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia确定的HCDR2包含与SEQ ID NO:4、17、44、56、67、78、91、120或85的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR2 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO:4, 17, 44, 56, 67, 78, 91, 120 or 85, or the HCDR2 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, 17, 44, 56, 67, 78, 91, 120 or 85.
在一些实施方案中，基于Kabat确定的HCDR2包含SEQ ID NO:7、20、31、37、47、59、69、81、94、103、111、123、98或108的氨基酸序列，或由所述氨基酸序列组成，或者基于Kabat确定的HCDR2包含与SEQ ID NO:7、20、31、37、47、59、69、81、94、103、111、123、98或108的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR2 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:7, 20, 31, 37, 47, 59, 69, 81, 94, 103, 111, 123, 98 or 108, or the HCDR2 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7, 20, 31, 37, 47, 59, 69, 81, 94, 103, 111, 123, 98 or 108.
在一些实施方案中，基于Chothia确定的HCDR3包含SEQ ID NO:5、18、29、45、57、68、79、92、102、121或86的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia确定的HCDR3包含与SEQ IDN O:5、18、29、45、57、68、79、92、102、121或86的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR3 determined based on Chothia comprises or consists of the amino acid sequence of SEQ ID NO: 5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86, or the HCDR3 determined based on Chothia comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86.
在一些实施方案中，基于Kabat确定的HCDR3包含SEQ ID NO:5、18、29、45、57、68、79、92、102、121或86的氨基酸序列，或由所述氨基酸序列组成，或者基于Kabat确定的HCDR3包含与SEQ ID NO:5、18、29、45、57、68、79、92、102、121或86的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the HCDR3 determined based on Kabat comprises or consists of the amino acid sequence of SEQ ID NO:5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86, or the HCDR3 determined based on Kabat comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:5, 18, 29, 45, 57, 68, 79, 92, 102, 121 or 86.
在一些实施方案中，基于Chothia和Kabat(Chothia&Kabat)确定的LCDR1包含SEQ ID NO:9、21、48、60、70、82、95、104、112或124的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia和Kabat(Chothia&Kabat)确定的LCDR1包含与SEQ ID NO:9、21、48、60、70、82、95、104、112或124的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。 In some embodiments, LCDR1 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence of SEQ ID NO: 9, 21, 48, 60, 70, 82, 95, 104, 112 or 124, or consists of the amino acid sequence, or LCDR1 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9, 21, 48, 60, 70, 82, 95, 104, 112 or 124.
在一些实施方案中，基于Chothia和Kabat(Chothia&Kabat)确定的LCDR2包含SEQ ID NO:10、22、49、61、71、83、96、105、113、125或52的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia和Kabat(Chothia&Kabat)确定的LCDR2包含与SEQ ID NO:10、22、49、61、71、83、96、105、113、125或52的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, LCDR2 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence of SEQ ID NO: 10, 22, 49, 61, 71, 83, 96, 105, 113, 125 or 52, or consists of the amino acid sequence, or LCDR2 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, 22, 49, 61, 71, 83, 96, 105, 113, 125 or 52.
在一些实施方案中，基于Chothia和Kabat(Chothia&Kabat)确定的LCDR3包含SEQ ID NO:11、23、32、38、50、62、72、84、97、106、114或12的氨基酸序列，或由所述氨基酸序列组成，或者基于Chothia和Kabat(Chothia&Kabat)确定的LCDR3包含与SEQ ID NO:11、23、32、38、50、62、72、84、97、106、114或12的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, the LCDR3 determined based on Chothia and Kabat (Chothia & Kabat) comprises or consists of the amino acid sequence of SEQ ID NO: 11, 23, 32, 38, 50, 62, 72, 84, 97, 106, 114 or 12, or the LCDR3 determined based on Chothia and Kabat (Chothia & Kabat) comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11, 23, 32, 38, 50, 62, 72, 84, 97, 106, 114 or 12.
在一些实施方案中，本发明的抗体重链恒定区为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。在一些实施方案中，本发明的抗体轻链恒定区为lambda或Kappa轻链恒定区，优选的Kappa轻链恒定区。在一些实施方案中，所述抗体或其抗原结合片段是IgG1形式的抗体或其抗原结合片段，并且包含人Kappa轻链恒定区。In some embodiments, the antibody heavy chain constant region of the present invention is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1. In some embodiments, the antibody light chain constant region of the present invention is a lambda or kappa light chain constant region, preferably a kappa light chain constant region. In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment in the form of IgG1 and comprises a human kappa light chain constant region.
在一些优选的实施方案中，本发明的抗体重链恒定区In some preferred embodiments, the heavy chain constant region of the antibody of the present invention is
(i)包含与选自SEQ ID NO:63的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 63;
(ii)包含选自SEQ ID NO:63的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:63; or
(iii)包含与选自SEQ ID NO:63的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成；或者(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 63; or
(iv)由下述核酸序列所编码，所述核酸序列(iv) is encoded by the following nucleic acid sequence,
(a)包含与选自SEQ ID NO:73的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(a) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO:73;
(b)包含选自SEQ ID NO:73的氨基酸序列或由所述氨基酸序列组成；或者(b) comprises or consists of an amino acid sequence selected from SEQ ID NO:73; or
(c)包含与选自SEQ ID NO:73的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(c) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 73.
在一些实施方案中，所述氨基酸改变发生在Fc区。在一些实施方案中，本发明的Fc区In some embodiments, the amino acid changes occur in the Fc region. In some embodiments, the Fc region of the present invention
(i)包含与选自SEQ ID NO:8的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO:8;
(ii)包含选自SEQ ID NO:8的氨基酸序列或由所述氨基酸序列组成；(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 8;
(iii)包含与选自SEQ ID NO:8的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成；或者(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 8; or
(iv)由下述核酸序列所编码，所述核酸序列 (iv) is encoded by the following nucleic acid sequence,
(a)包含与选自SEQ ID NO:87的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(a) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO:87;
(b)包含选自SEQ ID NO:87的氨基酸序列或由所述氨基酸序列组成；或者(b) comprises or consists of an amino acid sequence selected from SEQ ID NO:87; or
(c)包含与选自SEQ ID NO:87的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(c) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:87.
在一些实施方案中，本发明的抗体轻链恒定区In some embodiments, the antibody light chain constant region of the invention
(i)包含与选自SEQ ID NO:64的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 64;
(ii)包含选自SEQ ID NO:64的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:64; or
(iii)包含与选自SEQ ID NO:64的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:64.
在本发明的一些具体实施方案中，本发明的抗FRα抗体或其抗原结合片段包含：In some specific embodiments of the present invention, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises:
1)如SEQ ID NO:1或13所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:2、24或25所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；1) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1 or 13, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, 24 or 25;
2)如SEQ ID NO:41、33或34所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42、39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；2) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 41, 33 or 34, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 42, 39, 40 or 51;
3)如SEQ ID NO:74、99、115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42、39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；3) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 74, 99, 115 or 116, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 42, 39, 40 or 51;
4)如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:2所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；4) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2;
5)如SEQ ID NO:14所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:15所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；5) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15;
6)如SEQ ID NO:26所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:27所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；6) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:26, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27;
7)如SEQ ID NO:35所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:36所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；7) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:35, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:36;
8)如SEQ ID NO:41所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42所示的VL中所含的三个互补决定区域LCDR1、 LCDR2和LCDR3；8) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:41, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:42. LCDR2 and LCDR3;
9)如SEQ ID NO:53所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:54所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；9) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:53, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:54;
10)如SEQ ID NO:65所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:66所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；10) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:65, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:66;
11)如SEQ ID NO:75所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:76所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；11) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:76;
12)如SEQ ID NO:88所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:89所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；12) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:88, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:89;
13)如SEQ ID NO:100所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:101所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；13) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 100, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 101;
14)如SEQ ID NO:109所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:110所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；14) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 109, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 110;
15)如SEQ ID NO:117所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:118所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；15) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 117, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 118;
16)如SEQ ID NO:74或99所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：42所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；16) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 74 or 99, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 42;
17)如SEQ ID NO:13所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:24或25所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；17) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 24 or 25;
18)如SEQ ID NO:33所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；18) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:33, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39, 40 or 51;
19)如SEQ ID NO:34所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；19) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:34, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39, 40 or 51;
20)如SEQ ID NO:115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；20) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 115 or 116, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 39, 40 or 51;
21)如SEQ ID NO:115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2 和HCDR3，和如SEQ ID NO:51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；21) three complementary determining regions HCDR1, HCDR2 contained in VH as shown in SEQ ID NO: 115 or 116 and HCDR3, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:51;
优选地，其中所述HCDR和所述LCDR根据Chothia或Kabat确定，或根据Chothia和Kabat确定；Preferably, wherein said HCDR and said LCDR are determined according to Chothia or Kabat, or according to Chothia and Kabat;
例如，所述HCDR根据Kabat方案确定，所述LCDR根据Kabat和Chothia组合方案(Chothia&Kabat)确定；For example, the HCDR is determined according to the Kabat scheme, and the LCDR is determined according to the Kabat and Chothia combined scheme (Chothia &Kabat);
例如，所述HCDR根据Chothia方案确定，所述LCDR根据Kabat和Chothia组合方案(Chothia&Kabat)确定。For example, the HCDR is determined according to the Chothia scheme, and the LCDR is determined according to the combined Kabat and Chothia scheme (Chothia & Kabat).
在本发明的一些具体实施方案中，本发明的抗FRα抗体或其抗原结合片段包含：互补决定区HCDR1、HCDR2和HCDR3，以及LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2和HCDR3基于Chothia方案确定，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成，且所述LCDR1、LCDR2和LCDR3基于Kabat和Chothia组合方案(Chothia&Kabat)，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成：
In some specific embodiments of the present invention, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises: complementary determining regions HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 are determined based on the Chothia scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs, and the LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combined scheme (Chothia & Kabat), and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs:
或者其中所述HCDR1、HCDR2和HCDR3基于Kabat方案确定，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成，且所述LCDR1、LCDR2和LCDR3基于Kabat和Chothia组合方案，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成：
Or wherein the HCDR1, HCDR2 and HCDR3 are determined based on the Kabat scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs, and the LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combined scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs:
在本发明的一些具体实施方案中，本发明的抗FRα抗体或其抗原结合片段包含VH和VL，其中VH和VL分别包含如下表中所列SEQ ID NO所示的氨基酸序列，或与下表中所列SEQ ID NO所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成：

In some specific embodiments of the present invention, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises VH and VL, wherein VH and VL respectively comprise the amino acid sequence shown in SEQ ID NO as listed in the following table, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO as listed in the following table, or consist of the amino acid sequence:

在优选的实施方案中，本发明提供抗FRα抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述抗体或其抗原结合片段包含的重链可变区和轻链可变区包含如下表SEQ ID NO所示的氨基酸序列或由所述氨基酸序列组成：

In a preferred embodiment, the present invention provides an anti-FRα antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprised by the antibody or the antigen-binding fragment thereof comprise or consist of the amino acid sequence shown in the following table SEQ ID NO:

在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (eg, in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.
在某些实施方案中，置换发生在抗体的CDR区。通常，获得的变体相对于亲本抗体在某些生物学特性方面(例如，增加的亲和力)具有修饰(例如，改善)和/或将具有亲本抗体的基本上保留的某些生物学特性。例如，通过异构化位点进行置换来消除抗体的分子异构化的潜在风险，例如，可以将CDR，例如轻链CDR，例如轻链CDR2中的D突变，例如突变为E，例如，将第56位的D突变为E；例如，在本发明的抗体的Pha3-HZ2-D/EG-VK中，轻链的CDR2中的D突变为E，D56E(Kabat)。In certain embodiments, the substitution occurs in the CDR region of the antibody. Generally, the variant obtained has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and/or will have certain biological properties that are substantially retained by the parent antibody. For example, the potential risk of molecular isomerization of the antibody is eliminated by substitution at the isomerization site, for example, a CDR, such as a light chain CDR, such as a D mutation in a light chain CDR2, for example, can be mutated to E, for example, the D at position 56 is mutated to E; for example, in the Pha3-HZ2-D/EG-VK of the antibody of the present invention, the D in the CDR2 of the light chain is mutated to E, D56E (Kabat).
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。In certain embodiments, it may be desirable to generate cysteine engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues.
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基 纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。In certain embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl Cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (e.g. glycerol), polyvinyl alcohol, and mixtures thereof.
在一些实施方案中，本发明的抗体是人源化的。人源化可以通过如下方式实现：将非人源的天然抗体重链可变区和轻链可变区中的一个或多个氨基酸残基，尤其是构架区序列，置换成来自人的常规抗体的可变区相应位置的残基。人源化抗体的方法在本领域中是熟知的。典型地，人源化置换以保持抗体的有利结合性质的方式进行。本领域熟知用于确定人源化抗体的生物学性质，例如结合亲和力等的试验，以确定和选择适宜的人源化残基突变或突变组合。In some embodiments, the antibodies of the present invention are humanized. Humanization can be achieved by replacing one or more amino acid residues in the heavy chain variable region and light chain variable region of a natural antibody of non-human origin, especially the framework region sequence, with residues at corresponding positions in the variable region of a conventional antibody from a person. Methods for humanizing antibodies are well known in the art. Typically, humanization substitutions are carried out in a manner that maintains the favorable binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, etc., are well known in the art to determine and select suitable humanized residue mutations or mutation combinations.
在一些实施方案中，可以通过包括如下步骤的方法，获得本发明人源化的抗体：In some embodiments, the humanized antibody of the present invention can be obtained by a method comprising the following steps:
①确定亲本抗体(例如来自杂交瘤制备的抗体)的CDR环结构；① Determine the CDR loop structure of the parent antibody (e.g., an antibody prepared from a hybridoma);
②在人种系序列数据库(例如IMGT数据库)为每个V/J区域找到最接近的同源序列；② Find the closest homologous sequence for each V/J region in the human germline sequence database (such as the IMGT database);
③筛选与重链最匹配的人种系以及最低量的回复突变，在一个实施方案中，人种系基因是IGVH/IGKV种系基因；将嵌合抗体的CDR区构建至人的构架区上；③ Screening for the human germline that best matches the heavy chain and the lowest amount of back mutations. In one embodiment, the human germline gene is the IGVH/IGKV germline gene; constructing the CDR region of the chimeric antibody onto the human framework region;
④使用序列和结构特征，确定构架区中起到维持CDR功能的氨基酸位置；④Use sequence and structural features to determine the amino acid positions in the framework region that maintain the CDR function;
⑤在确定为重要的序列位置进行回复突变(返回到输入氨基酸类型)，例如，轻链可变区可以包含Q79E和/或Y87F和/或F71Y，或在重链可变区可以包含A24G，例如在2F22-HZ1的轻链可变区Q79E和Y87F(Kabat编号)，Phage3-HZ1的重链可变区A24G(Kabat编号)和轻链可变区的F71Y(Kabat编号)；⑤ Perform back mutations (return to the input amino acid type) at the sequence positions determined to be important, for example, the light chain variable region may contain Q79E and/or Y87F and/or F71Y, or the heavy chain variable region may contain A24G, such as Q79E and Y87F (Kabat numbering) in the light chain variable region of 2F22-HZ1, A24G (Kabat numbering) in the heavy chain variable region of Phage3-HZ1, and F71Y (Kabat numbering) in the light chain variable region;
⑥优化风险位点的氨基酸，例如对异构化位点进行改变来消除异构化风险，例如可以将CDR，例如轻链CDR，例如轻链CDR2中的D突变，例如突变为E，例如，在本发明的抗体Phage3-HZ2-D/EG-VK的LCDR2的氨基酸D突变为E,，例如其中第56位氨基酸D突变为E(Kabat编号)；和⑥ Optimizing the amino acids at risk sites, such as changing the isomerization sites to eliminate the isomerization risk, for example, the D in the CDR, such as the light chain CDR, such as the light chain CDR2 can be mutated, for example, to E, for example, the amino acid D in LCDR2 of the antibody Phage3-HZ2-D/EG-VK of the present invention is mutated to E, for example, wherein the amino acid D at position 56 is mutated to E (Kabat numbering); and
⑦获得人源化抗体，任选地测序抗体序列。⑦ Obtain humanized antibodies and optionally sequence the antibody sequences.
在一些实施方案中，本发明的抗FRα抗体或其抗原结合片段还包括具有以下一个或多个特性的抗体或抗原结合片段：In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further include antibodies or antigen-binding fragments having one or more of the following properties:
(i)显示与本发明抗体对FRα相同或相似的结合亲和力和/或特异性；(i) showing the same or similar binding affinity and/or specificity to FRα as the antibodies of the present invention;
(ii)抑制(例如，竞争性抑制)本发明抗体与FRα的结合；(ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the present invention to FRα;
(iii)与本发明抗体结合相同或重叠的表位；(iii) binds to the same or overlapping epitope as an antibody of the invention;
(iv)与本发明抗体竞争结合FRα；(iv) competing with the antibody of the present invention for binding to FRα;
(v)具有本发明抗体的一个或多个生物学特性。(v) possess one or more biological properties of the antibodies of the invention.
如本文所述定义的，与参照抗体“结合相同或重叠表位的抗体”是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合， 反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。As defined herein, an "antibody that binds to the same or overlapping epitope as a reference antibody" refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks greater than 50%, 60%, 70%, 80%, 90% or 95% of the binding of that antibody to its antigen in a competition assay.
如本文所述定义的，与参照抗体竞争结合其抗原的抗体是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。众多类型的竞争性结合测定可用于确定一种抗体是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定、生物光干涉测定法(例如Fortebio)或表面等离子共振(Biacore)等。As defined herein, an antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, biointerferometry (e.g., Fortebio), or surface plasmon resonance (Biacore), etc.
如本文所述定义的，抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。As defined herein, an antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, a reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that has at least 50%, 60%, 70%, 80%, 90% or 95% or more of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.
在一些实施方案中，本发明的抗FRα抗体是IgG1形式的抗体或IgG2形式的抗体或IgG3形式的抗体或IgG4形式的抗体。In some embodiments, the anti-FRα antibody of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.
在一些实施方案中，抗FRα抗体是单克隆抗体。In some embodiments, the anti-FRα antibody is a monoclonal antibody.
在一些实施方案中，抗FRα抗体是嵌合抗体或人源化抗体。In some embodiments, the anti-FRα antibody is a chimeric antibody or a humanized antibody.
在一个实施方案中，本发明的抗FRα抗体还涵盖其抗体片段，优选地选自以下的抗体片段：抗原结合片段包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、双体抗体(diabody)、单域抗体(sdAb)、纳米抗体、sc(Fv)2。In one embodiment, the anti-FRα antibodies of the present invention also encompass antibody fragments thereof, preferably selected from the following antibody fragments: antigen-binding fragments including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single domain antibody (sdAb), nanobody, sc(Fv)2.
在某些实施方案中，抗FRα抗体分子处于双特异性或多特异性抗体分子形式。在一个实施方案中，双特异性抗体可以结合FRα蛋白的两种不同的表位。在一个实施方案中，双特异性抗体可以结合FRα结合位点和另一种蛋白质。在一些实施方案中，所述抗FRα抗体是双特异性抗体或多特异性抗体。In certain embodiments, the anti-FRα antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody can bind to two different epitopes of the FRα protein. In one embodiment, the bispecific antibody can bind to the FRα binding site and another protein. In some embodiments, the anti-FRα antibody is a bispecific antibody or a multispecific antibody.
III.本发明的核酸以及包含其的宿主细胞III. Nucleic acids of the present invention and host cells containing the same
在一方面，本发明提供了编码以上任何抗FRα抗体或其片段或其任一条链的核酸的核酸。In one aspect, the present invention provides nucleic acids encoding any of the above anti-FRα antibodies or fragments thereof or either chain thereof.
例如，本发明的核酸包含编码选自SEQ ID NO:1、2、14、15、26、27、35、36、41、42、53、54、65、66、75、76、88、89、100、101、109、110、117、118、13、24、25、33、34、39、40、51、74、99、115或116中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:1、2、14、15、26、27、35、36、41、42、53、54、65、66、75、76、88、89、100、101、109、110、117、118、13、24、25、33、34、39、40、51、74、99、115或116中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。如本领域技术人员明了的，因为密码子简并性， 每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。编码本发明分子的核酸序列可以采用本领域熟知的方法，例如通过从头固相DNA合成，或通过PCR扩增而产生。For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 14, 15, 26, 27, 35, 36, 41, 42, 53, 54, 65, 66, 75, 76, 88, 89, 100, 101, 109, 110, 117, 118, 13, 24, 25, 33, 34, 39, 40, 51, 74, 99, 115 or 116, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: NO:1, 2, 14, 15, 26, 27, 35, 36, 41, 42, 53, 54, 65, 66, 75, 76, 88, 89, 100, 101, 109, 110, 117, 118, 13, 24, 25, 33, 34, 39, 40, 51, 74, 99, 115 or 116 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of NO:1, 2, 14, 15, 26, 27, 35, 36, 41, 42, 53, 54, 65, 66, 75, 76, 88, 89, 100, 101, 109, 110, 117, 118, 13, 24, 25, 33, 34, 39, 40, 51, 74, 99, 115 or 116. As will be appreciated by those skilled in the art, due to codon degeneracy, Each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid phase DNA synthesis, or by PCR amplification.
在一方面，本发明提供编码以上任何抗体或其片段或其任一条链的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示人(和/或恒河猴)FRα抗原结合能力。例如，在一些实施方案中，所述编码重链和/或轻链的可变区的核酸在读框中与编码重链和/或轻链的恒定区的核酸可操作连接，从而当从适宜的表达载体表达时，产生编码所述抗体重链和/或的核酸。In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any of their chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can display human (and/or rhesus monkey) FRα antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and/or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and/or light chain, thereby generating nucleic acids encoding the antibody heavy chain and/or when expressed from a suitable expression vector.
为了便于生产和纯化，可以在抗体的重链和/或轻链的N端融合分泌性信号肽，和/或利于纯化的标签肽。To facilitate production and purification, a secretory signal peptide and/or a tag peptide that facilitates purification may be fused to the N-terminus of the heavy chain and/or light chain of the antibody.
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是pCDNA载体，例如pCDNA3.1。In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC). In one embodiment, the vector is a pCDNA vector, such as pCDNA3.1.
在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞(例如CHO-S或CHO-K)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在一个实施方案中，宿主细胞是原核的，例如是细菌，例如大肠杆菌。In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, e.g., bacteria, e.g., Escherichia coli.
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293、293F或293T细胞)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括CHO-S细胞或CHO-K等；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。本领域已知适合产生抗体的哺乳动物宿主细胞系。In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungi and yeast strains that have been "humanized" in the glycosylation pathway lead to the production of antibodies with partial or complete human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO-S cells or CHO-K, etc.; and myeloma cell lines such as Y0, NS0 and Sp2/0. Mammalian host cell lines suitable for producing antibodies are known in the art.
IV.本发明的抗体分子的生产和纯化IV. Production and purification of antibody molecules of the invention
在一个实施方案中，本发明提供制备抗FRα抗体或其片段(优选的抗原结合片段)的方法，其中所述方法包括在适于表达编码所述抗体或其片段(优选的抗原结合片段)或其任一条或两条链的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体或其片段(优选地抗原结合片段)。在某个实施方案中，所述方法还包括从宿主细胞回收抗FRα抗体或其片段(优选地抗原结合片段)。In one embodiment, the present invention provides a method for preparing an anti-FRα antibody or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or a fragment thereof (preferably an antigen-binding fragment) or any one or both chains thereof, and optionally isolating the antibody or a fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-FRα antibody or a fragment thereof (preferably an antigen-binding fragment) from the host cell.
可以将编码本发明抗体的多肽链的多核苷酸插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。可以使用本领域技术人员熟知的方法来构建表达载体。一旦已经制备了用于表达的包含本发明的一种或多种核酸分子的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and/or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
如本文所述制备的抗体可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析例如Protein A、尺寸排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。 Antibodies prepared as described herein can be purified by known in the art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography such as Protein A, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.
可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。The purity of the antibody molecules of the invention can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
V.测定法V. Assay
可以通过本领域中已知的多种测定法对本文中提供的抗FRα抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。The anti-FRα antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.
一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如生物膜薄层干涉技术、ELISA，等来进行。可使用本领域已知方法来测定对FRα的结合，本文中公开了例示性方法。在一些实施方案中，使用放射性免疫测定(RIA)或生物膜薄层干涉测定法(BLI)或电化学发光(ECL)或表面等离子体共振法(SPR)或流式细胞术(FACS)测量。On the one hand, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as thin-layer interferometry, ELISA, etc. Binding to FRα can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or thin-layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used for measurement.
本发明还提供了用于鉴定具有生物学活性的抗FRα抗体的测定法。生物学活性选自本发明的抗体的性质，可以包括例如结合FRα(例如结合人和/或恒河猴FRα)、细胞内吞活性或包含所述抗体的分子的杀伤活性等。The present invention also provides an assay for identifying anti-FRα antibodies with biological activity. The biological activity is selected from the properties of the antibodies of the present invention, and may include, for example, binding to FRα (e.g., binding to human and/or rhesus monkey FRα), endocytic activity, or killing activity of molecules comprising the antibody, etc.
例如，对于本发明的抗体分子对FRα或表达FRα的细胞的结合活性，可以通过本领域已知的方法，例如Fortebio、流式细胞术、Octet或等离子共振(Biacore)等，或本文实施例公开的示例性方法测定。For example, the binding activity of the antibody molecules of the present invention to FRα or cells expressing FRα can be determined by methods known in the art, such as Fortebio, flow cytometry, Octet or Biacore, or the exemplary methods disclosed in the Examples herein.
例如，对于本发明的抗体分子的细胞(例如FRα阳性细胞)内吞活性，可以通过本领域已知的方法，例如流式细胞术，或本文实施例中公开的示例性方法测定。For example, the cellular (eg, FRα-positive cell) endocytosis activity of the antibody molecule of the present invention can be determined by methods known in the art, such as flow cytometry, or the exemplary methods disclosed in the Examples herein.
例如，对于包含本发明的抗体分子的杀伤活性，可以通过本领域已知的方法，例如细胞杀伤实验，如体外细胞杀伤实验，或本文实施例中公开的示例性方法测定。For example, the killing activity of the antibody molecules of the present invention can be determined by methods known in the art, such as cell killing assays, such as in vitro cell killing assays, or the exemplary methods disclosed in the Examples herein.
例如，对于本发明的抗体分子的药物动力学性质，可以通过本领域已知的方法，例如本文实施例公开的示例性方法测定。For example, the pharmacokinetic properties of the antibody molecules of the present invention can be determined by methods known in the art, such as the exemplary methods disclosed in the Examples herein.
供任何上述体外测定法使用的细胞包括天然表达FRα或经改造而表达FRα细胞系。此类细胞还包括表达FRα和并非正常情况下表达FRα的编码FRαDNA转染的细胞系。Cells for use in any of the above in vitro assays include cell lines that naturally express FRα or are engineered to express FRα. Such cells also include cell lines that express FRα and are transfected with FRα-encoding DNA that does not normally express FRα.
可以理解的是，能够使用本发明的免疫缀合物替换或补充抗FRα抗体来进行任何上述测定法。It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to the anti-FRα antibody.
可以理解的是，能够使用抗FRα抗体和别的活性剂组合来进行任何上述测定法。It will be appreciated that any of the above assays can be performed using an anti-FRα antibody in combination with an additional active agent.
VI.免疫缀合物VI. Immunoconjugates
在一些实施方案中，本发明提供了免疫缀合物，其包含本文中提供的任何抗FRα抗体和其它物质，例如治疗剂或标记。在一些实施方案中，治疗剂可以是适合与FRα形成免疫缀合物的治疗剂，例如免疫调节剂，例如抗炎剂或免疫抑制剂。在一些实施方案中，治疗剂选自化疗剂、细胞毒性剂、细胞因子、小分子药物、免疫调节剂(例如免疫抑制剂)或其他抗体。In some embodiments, the present invention provides an immunoconjugate comprising any anti-FRα antibody provided herein and other substances, such as therapeutic agents or markers. In some embodiments, the therapeutic agent can be a therapeutic agent suitable for forming an immunoconjugate with FRα, such as an immunomodulator, such as an anti-inflammatory agent or an immunosuppressant. In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a cytokine, a small molecule drug, an immunomodulator (e.g., an immunosuppressant) or other antibodies.
在一些实施方案中，所述免疫缀合物用于预防或治疗FRα相关疾病，例如肿瘤或癌症。 In some embodiments, the immunoconjugate is used to prevent or treat FRα-related diseases, such as tumors or cancer.
VII.药物组合物和药物制剂VII. Pharmaceutical compositions and pharmaceutical preparations
在一些实施方案中，本发明提供包含本文所述的任何抗FRα抗体或其片段(优选地其抗原结合片段)或其免疫缀合物的组合物，优选地组合物为药物组合物或药物制剂。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的抗FRα抗体或其片段或其免疫缀合物，以及一种或多种其它治疗剂(例如如化疗剂、细胞因子、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂))的组合。本发明还包括包含编码抗FRα抗体的多核苷酸的组合物(包括药物组合物或药物制剂)。In some embodiments, the present invention provides a composition comprising any anti-FRα antibody or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof as described herein, preferably the composition is a pharmaceutical composition or a pharmaceutical preparation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-FRα antibody or fragment thereof or an immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents (e.g., such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs or immunomodulators (e.g., immunosuppressants)). The present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising a polynucleotide encoding an anti-FRα antibody.
在某些实施方案中，组合物包含一种或多种结合FRα的抗体或其片段，或一种或多种编码一种或多种抗FRα的抗体或其片段的多核苷酸。这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。In certain embodiments, the composition comprises one or more antibodies or fragments thereof that bind to FRα, or one or more polynucleotides encoding one or more anti-FRα antibodies or fragments thereof. These compositions may also comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。For the use and purposes of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
可以通过将具有所需纯度的本发明的抗体与一种或多种任选的药用辅料混合来制备包含本文所述的抗体的药物制剂，优选地以冻干制剂或水溶液的形式。Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing the antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂，例如如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。所述活性成分以对于目的用途有效的量合适地组合存在。The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activity that will not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants). The active ingredient is suitably combined in an amount effective for the intended use.
VIII.药物组合和药盒VIII. Pharmaceutical Combinations and Kits
在一些实施方案中，本发明还提供了药物组合或药物组合产品，其包含本发明的抗FRα抗体或其片段(优选的抗原结合片段)，或其免疫缀合物，以及一种或多种其它治疗剂(例如如化疗剂、细胞因子、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂)。In some embodiments, the present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising an anti-FRα antibody or a fragment thereof (preferably an antigen-binding fragment) of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants).
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式。由此可以依据给药方案或药物施用间隔提供剂量单元。Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, so that the dosage unit can be provided according to the dosage regimen or the interval of drug administration.
在一个实施方案中，本发明的成套药盒在同一包装内包含：In one embodiment, the kit of parts of the present invention comprises in the same package:
-含有包含抗FRα抗体或其片段的药物组合物的第一容器；- a first container containing a pharmaceutical composition comprising an anti-FRα antibody or a fragment thereof;
-含有包含其它治疗剂如化疗剂、细胞因子、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂)的药物组合物的第二容器。- A second container containing a pharmaceutical composition comprising another therapeutic agent, such as a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a vaccine, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).
在一些实施方案中，所述组合产品用于预防或治疗FRα相关疾病，例如肿瘤或癌症。 In some embodiments, the combination product is used to prevent or treat FRα-related diseases, such as tumors or cancer.
IX.用途和方法IX. Purpose and Method
本发明一方面提供了在受试者中治疗FRα相关疾病的方法，包括向受试者施用有效量的本发明的抗FRα的抗体或其抗原结合片段、免疫缀合物、药物组合物或组合产品。In one aspect, the present invention provides a method for treating a FRα-related disease in a subject, comprising administering to the subject an effective amount of the anti-FRα antibody or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition or combination product of the present invention.
在一些实施方案中，本文所述的FRα相关疾病包括肿瘤，例如癌症。癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，癌症可以是实体肿瘤或血液肿瘤。在一些实施方案中，所述癌症是例如上皮细胞癌，例如肺癌(例如非小细胞肺癌)、口腔癌(如口腔上皮癌)、卵巢癌、乳腺癌、间质瘤、子宫内膜癌或宫颈癌。In some embodiments, the FRα-related diseases described herein include tumors, such as cancer. The cancer may be in the early, middle or late stages or may be metastatic cancer. In some embodiments, the cancer may be a solid tumor or a blood tumor. In some embodiments, the cancer is, for example, an epithelial cell cancer, such as lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumors, endometrial cancer, or cervical cancer.
在一个实施方案中，所述肿瘤是指在个体的肿瘤组织或肿瘤细胞中高表达FRα的肿瘤。在一个实施方案中，所述肿瘤是指在个体的肿瘤组织或肿瘤细胞中，例如与个体的相邻正常组织或正常细胞(例如组织中的正常细胞)或健康个体的相同组织或其中的细胞相比，FRα的蛋白质水平(例如表达)升高，或FRα的核酸水平升高。In one embodiment, the tumor refers to a tumor that highly expresses FRα in the tumor tissue or tumor cells of the individual. In one embodiment, the tumor refers to an increase in the protein level (e.g., expression) of FRα, or an increase in the nucleic acid level of FRα, in the tumor tissue or tumor cells of the individual, for example, compared with the adjacent normal tissue or normal cells of the individual (e.g., normal cells in the tissue) or the same tissue or cells therein of a healthy individual.
在其他方面，本发明提供抗FRα抗体或其片段在生产或制备药物中的用途，所述药物用于治疗本文提及的相关疾病或病症。In other aspects, the present invention provides the use of an anti-FRα antibody or a fragment thereof in the production or preparation of a medicament for treating the relevant diseases or disorders mentioned herein.
在一些实施方案中，本发明的抗体或抗体片段或免疫缀合物或组合物或产品会延迟病症和/或与病症相关的症状的发作。In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of a disorder and/or symptoms associated with a disorder.
在一些实施方案中，本文所述的预防或治疗方法还包括向所述受试者或个体组合施用本文公开的抗体分子或药物组合物或免疫缀合物，以及一种或多种其它疗法，例如治疗方式和/或其它治疗剂。In some embodiments, the preventive or therapeutic methods described herein further comprise administering to the subject or individual an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents.
在一些实施方案中，治疗方式包括外科手术、放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的照射)或聚焦照射)等。In some embodiments, treatment modalities include surgery, radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed to a preselected target or organ), or focused irradiation), etc.
在一些实施方案中，治疗剂选自化疗剂、细胞因子、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂)等。In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytokine, a cytotoxic agent, other antibodies, a vaccine, a small molecule drug, or an immunomodulator (eg, an immunosuppressant), among others.
在一些实施方案中，本文中描述的抗体可以与其他抗体组合，用于分别施用，例如，分别作为单独的抗体分别施用，或连接时(例如作为双特异性或多特异性抗体分子)施用。In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., separately as separate antibodies, or when linked (e.g., as a bispecific or multispecific antibody molecule).
此类组合疗法涵盖组合施用(例如两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的治疗剂和/或药剂之前，同时，和/或之后发生本发明的抗体的施用。Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or in separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agents and/or agents.
本发明的抗FRα抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)可以通过任何合适的方法给药，包括肠胃外给药，并且，如果局部治疗需要，病灶内给药。肠胃外注射或输注包括肌内、静脉内、动脉内、腹膜内或皮下注射或输注。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。The anti-FRα antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending to some extent on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
为了预防或治疗疾病，本发明的抗FRα抗体或其片段(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用 还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，施用模式；施用制剂的生物利用率特征；选择的给药方案；任何伴随疗法的使用和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。For the prevention or treatment of disease, the appropriate dosage of the anti-FRα antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, administration for preventive purposes, Whether administered for therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of any concomitant therapy and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
在其他方面，本发明提供本发明的抗FRα抗体或其片段或包含其的免疫缀合物或组合物在生产或制备药物中的用途，所述药物用于本文所述的用途，例如用于预防或治疗本文提及的相关疾病或病症。In other aspects, the present invention provides use of the anti-FRα antibody or fragment thereof, or an immunoconjugate or composition comprising the same, of the present invention in the production or preparation of a medicament for the uses described herein, such as for preventing or treating the relevant diseases or disorders mentioned herein.
在一些实施方案中，抗FRα抗体或其片段(以及包含其的免疫缀合物、组合物、药物组合物、制剂等)还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。In some embodiments, the anti-FRα antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
X.用于诊断和检测的方法和组合物X. Methods and compositions for diagnosis and detection
在一个方面，本发明还涉及对本发明抗体或其抗原结合片段的用于诊断和检测的方法和包含其的用于诊断和检测的组合物。In one aspect, the present invention also relates to methods for diagnosis and detection of the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosis and detection comprising the same.
在某些实施方案中，本文中提供的任何抗FRα抗体或其抗原结合片段可以用于检测FRα在生物样品中的存在。In certain embodiments, any of the anti-FRα antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of FRα in a biological sample.
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自肿瘤组织或癌症组织。The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is other liquid samples of blood, serum or biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from tumor tissue or cancer tissue.
在一个实施方案中，提供用于诊断或检测方法的抗FRα抗体。In one embodiment, an anti-FRα antibody for use in a method of diagnosis or detection is provided.
在另一个方面中，提供检测FRα在生物样品中的存在的方法。在某些实施方案中，所述方法包含检测FRα蛋白在生物样品中的存在。在某些实施方案中，FRα是人FRα或恒河猴FRα。在某些实施方案中，所述方法包括将生物样品与如本文所述的抗FRα抗体在允许抗FRα抗体与FRα结合的条件下接触，并检测在抗FRα抗体和FRα之间是否形成复合物。复合物的形成表示存在FRα。该方法可以是体外或体内方法。在一个实施方案中，抗FRα抗体被用于选择适合利用抗FRα抗体的治疗的受试者，例如其中FRα是用于选择所述受试者的生物标记物。In another aspect, a method for detecting the presence of FRα in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of FRα protein in a biological sample. In certain embodiments, FRα is human FRα or rhesus monkey FRα. In certain embodiments, the method comprises contacting the biological sample with an anti-FRα antibody as described herein under conditions that allow the anti-FRα antibody to bind to FRα, and detecting whether a complex is formed between the anti-FRα antibody and FRα. The formation of a complex indicates the presence of FRα. The method can be an in vitro or in vivo method. In one embodiment, an anti-FRα antibody is used to select a subject suitable for treatment with an anti-FRα antibody, for example, wherein FRα is a biomarker for selecting the subject.
在某些实施方案中，提供检测样品中FRα的方法，所述方法包括In certain embodiments, a method for detecting FRα in a sample is provided, the method comprising
(a)将样品与本发明的抗FRα抗体或其抗原结合片段接触；和(a) contacting a sample with the anti-FRα antibody or antigen-binding fragment thereof of the present invention; and
(b)检测抗FRα抗体或其抗原结合片段和FRα间的复合物的形成；任选地，抗FRα抗体是被可检测地标记的。(b) detecting formation of a complex between the anti-FRα antibody or antigen-binding fragment thereof and FRα; optionally, the anti-FRα antibody is detectably labeled.
在某些实施方案中，提供标记的抗体或其片段。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。In certain embodiments, labeled antibodies or fragments thereof are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detected moieties, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions.
在本文中提供的一些实施方案中，样品是在用本发明的抗体或其片段治疗之前获得的。在一些实施方案中，样品是在用其他疗法之前获得的。在一些实施方案中，样品是在用其他疗法治疗过程中，或者用其他疗法治疗后获得的。 In some embodiments provided herein, the sample is obtained before treatment with an antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained before other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies.
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测FRα。In some embodiments, FRα is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验FRα的存在，因而确定FRα值，将FRα值与对照值(例如健康个体或正常组织中的值)比较，并且如果FRα值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的抗体或其片段，因而治疗所述疾病。In some embodiments, a method for treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of FRα, thereby determining the FRα value, comparing the FRα value with a control value (e.g., a value in a healthy individual or normal tissue), and if the FRα value is greater than the control value, administering to the subject a therapeutically effective amount of an antibody or fragment thereof of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验FRα的存在，因而确定FRα值，将FRα值与对照值(例如正常个体中的值)比较，并且如果FRα值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的抗体或其片段或包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等，因而治疗所述疾病。In some embodiments, a method for treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of FRα, thereby determining the FRα value, comparing the FRα value with a control value (e.g., a value in a normal individual), and if the FRα value is greater than the control value, administering to the subject a therapeutically effective amount of the antibody or fragment thereof described in the present invention, or an immunoconjugate, composition, pharmaceutical composition, preparation, combination product, etc. comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art.
实施例：Example:
实施例1：鼠源单克隆抗体的筛选Example 1: Screening of mouse monoclonal antibodies
1.1骨髓瘤细胞培养1.1 Myeloma cell culture
骨髓瘤细胞P3X63Ag8.653(南京科佰公司，货号CBP60876)使用含有20ug/mL 8-氮鸟嘌呤(Merck#134-58-7)的培养基连续传代，在5％CO2，37℃细胞培养箱中培养。Myeloma cells P3X63Ag8.653 (Nanjing Kebai Company, catalog number CBP60876) were serially passaged using a culture medium containing 20 ug/mL 8-azaguanine (Merck #134-58-7) and cultured in a 5% CO 2 , 37° C. cell culture incubator.
1.2小鼠免疫1.2 Immunization of mice
使用商业化重组人FRα-his蛋白(近岸公司目录号C784)按照文献所述常规方法(Lonberg,N.,et al.,nature 368(1994)856-859；Fishwild,D.M.,et al.,Nat.Biotechnol.14(1996)845-851 and WO 98/24884)对10只Balb/c小鼠进行免疫。对每只小鼠使用重组人FRα进行4次皮下(SC)免疫。对于第一次免疫，将100ul(75ug)的重组人FRα溶液与100ul的完全弗氏佐剂混合，对于其它免疫，使用100ul(75ug)的重组人FRα溶液与100ul不完全弗氏佐剂混合。Ten Balb/c mice were immunized using commercial recombinant human FRα-his protein (Nearshore Company catalog number C784) according to the conventional method described in the literature (Lonberg, N., et al., nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884). Each mouse was immunized subcutaneously (SC) 4 times with recombinant human FRα. For the first immunization, 100ul (75ug) of recombinant human FRα solution was mixed with 100ul of complete Freund's adjuvant, and for other immunizations, 100ul (75ug) of recombinant human FRα solution was mixed with 100ul of incomplete Freund's adjuvant.
1.3抗原特异性ELISA检测小鼠血清滴度1.3 Antigen-specific ELISA to detect mouse serum titers
通过抗原特异性ELISA来确定免疫小鼠血清中的抗FRα效价。将浓度为1ug/ml的FRα溶液包被在96孔板中，每孔100ul，4℃过夜孵育。然后洗去包被液，使用封闭液(protein-free blocking buffer)加入每孔，室温静置孵育2小时。将小鼠血清在PBSA(含1％BSA的PBS溶液)中预稀释100倍，并1:2连续稀释10个稀释梯度。洗去包被液后，将稀释的血清加入板孔中在室温孵育1个小时。用PBST洗涤96孔板后加入100ul/孔1/30000稀释的二抗(HRP标记的羊抗鼠IgG(Jackson#515-035-003))室温静置孵育45分钟，PBST洗涤三遍，加入室温平衡的TMB 20ul/孔，室温孵育10分钟，在650nm测量吸光度(图1)。Antigen-specific ELISA was used to determine the anti-FRα titer in the sera of immunized mice. FRα solution with a concentration of 1ug/ml was coated in a 96-well plate, 100ul per well, and incubated overnight at 4°C. The coating solution was then washed off, and a blocking buffer (protein-free blocking buffer) was added to each well and incubated at room temperature for 2 hours. Mouse serum was pre-diluted 100 times in PBSA (PBS solution containing 1% BSA) and diluted 10 times in a 1:2 dilution gradient. After washing off the coating solution, the diluted serum was added to the plate wells and incubated at room temperature for 1 hour. After washing the 96-well plate with PBST, 100ul/well of a 1/30000 diluted secondary antibody (HRP-labeled goat anti-mouse IgG (Jackson #515-035-003)) was added and incubated at room temperature for 45 minutes, washed three times with PBST, and 20ul/well of TMB equilibrated at room temperature was added. Incubated at room temperature for 10 minutes, and the absorbance was measured at 650nm (Figure 1).
1.4小鼠的加强免疫 1.4 Booster immunization of mice
对抗FRα的血清效价大于1:100000的小鼠，在融合前4天，用溶于100ul PBS的100ug FRα溶液对小鼠进行另外的腹腔注射(IP)加强免疫一次。For mice with anti-FRα serum titers greater than 1:100,000, they were given an additional intraperitoneal (IP) booster immunization 4 days before fusion with 100 μg FRα solution dissolved in 100 μl PBS.
实施例2抗人FRα抗体的制备和表征Example 2 Preparation and Characterization of Anti-Human FRα Antibodies
2.1过表达人/猴FRα的HEK293细胞和CHO细胞株制备：2.1 Preparation of HEK293 cells and CHO cell lines overexpressing human/monkey FRα:
将人FRα的ORF(NCBI：NM_000802.3)和恒河猴FRα的ORF(NCBI：NM_001194647.3)分别构建插入pcDNA5/FRT载体(Invitrogen#V790-20)的多克隆位点中，依照Flp-In系统(Invitrogen，货号K6010)的操作说明，将构建好的重组质粒用lipofectamine3000试剂转染Flp-In HEK293细胞(Invitrogen公司#R758–07)和Flp-In CHO细胞(Invitrogen公司#R750–07)，转染48小时后，使用100μg/mL的Hygromycin进行筛选培养，10天后使用流式细胞仪将细胞进行单克隆分选，选择生长良好的单克隆使用特异性结合FRα的抗体进行鉴定并扩大培养，获得过表达hFRα的CHO细胞(命名为CHO-hFOLR1)、HEK293细胞(命名为HEK293-hFOLR1或HEK293-FRα)和过表达恒河猴FRα的CHO细胞(命名为CHO-cynoFOLR1)，用于细胞学实验。The ORF of human FRα (NCBI: NM_000802.3) and rhesus monkey FRα (NCBI: NM_001194647.3) were respectively constructed and inserted into the multiple cloning site of pcDNA5/FRT vector (Invitrogen #V790-20). According to the operating instructions of Flp-In system (Invitrogen, cat. no. K6010), the constructed recombinant plasmids were transfected into Flp-In HEK293 cells (Invitrogen company #R758–07) and Flp-In CHO cells ( #R750–07 from Invitrogen), 48 hours after transfection, 100 μg/mL Hygromycin was used for screening and culture, 10 days later, the cells were sorted for monoclonal separation using a flow cytometer, and monoclonal clones with good growth were selected and identified using antibodies that specifically bind to FRα and expanded for culture, thus obtaining CHO cells overexpressing hFRα (named CHO-hFOLR1), HEK293 cells (named HEK293-hFOLR1 or HEK293-FRα), and CHO cells overexpressing rhesus monkey FRα (named CHO-cynoFOLR1) for cytological experiments.
2.2抗人FRα杂交瘤产生及检测2.2 Anti-human FRα hybridoma production and detection
取实施例1获得的小鼠脾脏，将分离得到的脾细胞与骨髓瘤细胞P3X63Ag8.653使用电融合方法进行融合。将融合后的杂交瘤细胞接种于384孔板中，培养14天后，以人FRα-His重组蛋白(1ug/ml，pH9.6，0.1M NaHCO3)包被酶标板，4℃过夜孵育；用4％脱脂奶粉-PBS封闭，37℃，2小时孵育；用PBST(0.05％Tween20-PBS)洗三遍，加入杂交瘤克隆培养上清，37℃孵育1小时。经PBST(0.05％Tween20-PBS)洗三遍，加入HRP-羊抗小鼠IgG(Fcγ)(Jackson#515-035-071)，1:20000稀释，37℃孵育1小时；再经PBST(0.05％Tween20-PBS)洗五遍，加入TMB显色液，避光显色10min，酶标仪读A650光吸收值。(图2)Take the mouse spleen obtained in Example 1, and fuse the separated spleen cells with myeloma cells P3X63Ag8.653 using the electrofusion method. The fused hybridoma cells were inoculated in a 384-well plate, cultured for 14 days, coated with human FRα-His recombinant protein (1ug/ml, pH9.6, 0.1M NaHCO3), incubated at 4°C overnight; blocked with 4% skim milk powder-PBS, incubated at 37°C for 2 hours; washed three times with PBST (0.05% Tween20-PBS), added with hybridoma clone culture supernatant, and incubated at 37°C for 1 hour. After washing three times with PBST (0.05% Tween20-PBS), HRP-goat anti-mouse IgG (Fcγ) (Jackson #515-035-071) was added, diluted 1:20000, and incubated at 37°C for 1 hour; then washed five times with PBST (0.05% Tween20-PBS), TMB colorimetric solution was added, color was developed for 10 minutes in the dark, and the A650 light absorption value was read with an enzyme reader. (Figure 2)
使用96孔板检测过表达hFRα的CHO细胞(CHO-hFOLR1)的流式结合能力，每个检测孔使用2×105个细胞，加入杂交瘤上清20ul。4℃孵育1小时后，用PBS清洗；加入稀释1000倍的FITC标记抗人抗体的二抗(Jackson#109-095-098)，100mL/孔重悬细胞，4℃孵育1小时后，用PBS清洗，在流式细胞分析仪上分析(图3)。以同样的方法，使用过表达cyno FRα的CHO细胞(CHO-cynoFOLR1)，对候选克隆的上清做流式分析筛选。经检测鉴定，共获得多个分泌阳性FRα抗体，同时识别猴FRα分子的杂交瘤克隆。(图4)The flow cytometry binding ability of CHO cells (CHO-hFOLR1) overexpressing hFRα was detected using a 96-well plate. 2×10 5 cells were used in each test well, and 20ul of hybridoma supernatant was added. After incubation at 4°C for 1 hour, the cells were washed with PBS; a 1000-fold diluted FITC-labeled anti-human antibody secondary antibody (Jackson #109-095-098) was added, and the cells were resuspended at 100mL/well. After incubation at 4°C for 1 hour, the cells were washed with PBS and analyzed on a flow cytometer (Figure 3). In the same way, CHO cells (CHO-cynoFOLR1) overexpressing cyno FRα were used to perform flow cytometry screening on the supernatant of candidate clones. After detection and identification, a total of multiple hybridoma clones that secreted positive FRα antibodies and recognized monkey FRα molecules were obtained. (Figure 4)
2.3亚克隆2.3 Subcloning
将结合实验筛选得到的特异性识别人FRα的杂交瘤细胞克隆，通过有限稀释的方法单细胞化，两轮亚克隆之后得到的每个杂交瘤细胞克隆只分泌一个抗体。The hybridoma cell clones that specifically recognize human FRα obtained by binding experiment screening were subcloned into single cells by limiting dilution method. After two rounds of subcloning, each hybridoma cell clone obtained secreted only one antibody.
2.4抗体轻链可变区IgG VL和重链可变区IgG VH序列的克隆2.4 Cloning of antibody light chain variable region IgG VL and heavy chain variable region IgG VH sequences
根据特异性结合实验结果，选择结合能力强的抗体克隆。杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物 (Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区VL和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007 Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。According to the results of the specific binding experiment, antibody clones with strong binding ability were selected. After the hybridoma cells were expanded and cultured, total RNA of the cells was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); 5× PrimeScript RT Master Mix (Takara) was used to reverse transcribe the total RNA of the hybridoma cells into cDNA; degenerate primers were used (Anke Krebber.1997) and Extaq PCR reagent (Takara) were used to amplify the antibody light chain variable region VL and heavy chain variable region VH sequences; the PCR amplification products were purified using a PCR clean-up Gel extraction kit (Macherey-Nagel); according to the instructions of the pClone007 Simple Vector Kit (Qingke Biotechnology Co., Ltd.), the amplified PCR products were connected to the T vector and transformed into Escherichia coli competent cells, and the monoclonal antibody variable region sequences were obtained by DNA sequencing after strain amplification and plasmid extraction.
2.5抗人FRα噬菌体展示文库的制备和抗体筛选2.5 Preparation of anti-human FRα phage display library and antibody screening
收集免疫小鼠骨髓B细胞。用Qiagen RNA提取试剂盒从B细胞中提取总RNA，用逆转录试剂盒(Takara#RR037A)利用随机引物逆转录得到cDNA，以该cDNA为模板，利用抗体可变区引物PCR扩增得到抗体重轻链可变区片段；经overlap PCR获得单链抗体scFv片段。Sfi I酶切后，克隆到自主构建的噬菌体质粒pBluescript SK/KS中。然后构建基于丝状噬菌体M13的小鼠scFv噬菌体展示文库，库容为6x108。B cells from the bone marrow of immunized mice were collected. Total RNA was extracted from B cells using a Qiagen RNA extraction kit, and reverse transcription was performed using a reverse transcription kit (Takara#RR037A) using random primers to obtain cDNA. The cDNA was used as a template and PCR amplified using antibody variable region primers to obtain antibody heavy and light chain variable region fragments; single-chain antibody scFv fragments were obtained by overlap PCR. After Sfi I digestion, the fragments were cloned into the self-constructed phage plasmid pBluescript SK/KS. Then, a mouse scFv phage display library based on filamentous phage M13 was constructed with a library capacity of 6x10 8 .
取10ug生物素化的人FRα重组蛋白，与SA磁珠4度孵育过夜。用PBS-吐温(0.5％v/v)和PBS进行5轮洗涤，加入到1ml噬菌体中，旋转混匀结合约10分钟。用PBS-吐温(0.5％v/v)和PBS进行15轮洗涤以除去未结合的噬菌体，洗涤后的磁珠用来感染对数期大肠杆菌TG1细菌，拯救以进行下一轮富集筛选。第二轮筛选将投入的抗原降为5ug，第三轮为2ug。经过三轮富集筛选后，各取每一轮的少量TG1大肠杆菌涂LB平板，37度过夜培养后挑取单克隆至96孔U型孔板中进行IPTG诱导表达，取上清进行ELISA检测筛选，ELISA阳性克隆经测序获得抗体可变区的序列。Take 10ug of biotinylated human FRα recombinant protein and incubate it with SA magnetic beads at 4 degrees overnight. Wash with PBS-Tween (0.5% v/v) and PBS for 5 rounds, add to 1ml of phage, rotate and mix to bind for about 10 minutes. Wash with PBS-Tween (0.5% v/v) and PBS for 15 rounds to remove unbound phage. The washed magnetic beads are used to infect logarithmic phase Escherichia coli TG1 bacteria and rescue for the next round of enrichment screening. The second round of screening will reduce the input antigen to 5ug, and the third round to 2ug. After three rounds of enrichment screening, take a small amount of TG1 Escherichia coli from each round to coat the LB plate, culture overnight at 37 degrees, pick a single clone to 96-well U-shaped plate for IPTG induction expression, take the supernatant for ELISA detection screening, and the ELISA-positive clones are sequenced to obtain the sequence of the antibody variable region.
2.6抗人FRα嵌合抗体的制备2.6 Preparation of anti-human FRα chimeric antibodies
将鼠源抗人FRα单克隆抗体的重链可变区编码核酸序列和公开发表的人单克隆抗体IgG1亚类的重链恒定区序列编码核酸序列(SEQ ID NO:73)拼接在一起，构建到哺乳动物细胞表达载体pcDNATM3.1(+)(Invitrogen#V790-20)中；将鼠源抗人FRα单克隆抗体的轻链可变区编码核酸序列和公开发表的人单克隆抗体κ亚类的轻链恒定区编码核酸序列(SEQ ID NO:87)拼接在一起，构建到哺乳动物细胞表达载体pcDNATM3.1(+)(Invitrogen#V790-20)中。构建好的抗人α嵌合抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用ProteinA纯化得到抗人FRα嵌合抗体蛋白。The heavy chain variable region encoding nucleic acid sequence of the mouse anti-human FRα monoclonal antibody and the publicly published heavy chain constant region encoding nucleic acid sequence of the human monoclonal antibody IgG1 subclass (SEQ ID NO: 73) were spliced together and constructed into the mammalian cell expression vector pcDNA TM 3.1 (+) (Invitrogen # V790-20); the light chain variable region encoding nucleic acid sequence of the mouse anti-human FRα monoclonal antibody and the publicly published light chain constant region encoding nucleic acid sequence of the human monoclonal antibody κ subclass (SEQ ID NO: 87) were spliced together and constructed into the mammalian cell expression vector pcDNA TM 3.1 (+) (Invitrogen # V790-20). The constructed heavy chain vector and light chain vector of the anti-human α chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Protein A to obtain the anti-human FRα chimeric antibody protein.
类似的构建对照抗体MIRV(US20200362029A1)。A similarly constructed control antibody MIRV (US20200362029A1) was constructed.
获得的嵌合抗体的可变区序列和CDR序列的SEQ ID NO如下表1。The SEQ ID NO of the variable region sequence and CDR sequence of the obtained chimeric antibody is shown in Table 1.
后文在提及这些嵌合抗体时，应用抗体编号或应用抗体编号-xiIgG，其均代表具有表1所示的抗体编号下的可变区和IgG1的重链恒定区和κ亚类轻链恒定区的嵌合抗体。When referring to these chimeric antibodies hereinafter, the antibody number or the antibody number -xiIgG will be used, which both represent chimeric antibodies having the variable region under the antibody number shown in Table 1 and an IgG1 heavy chain constant region and a κ subclass light chain constant region.
表1，具有如下编号的嵌合抗体的可变区和CDR序列。

Table 1, variable region and CDR sequences of chimeric antibodies with the following numbering.

2.7 FRα嵌合抗体的体外亲和力的动力学研究2.7 Kinetic study of in vitro affinity of FRα chimeric antibodies
利用Fortebio(BLITZ pro1.1.0.28)仪器分析嵌合抗体与抗原人FRα(近岸蛋白#C784)的结合动力学参数。测定前先将AHC生物探针(AHC，Satorius#18-5064)浸泡于PBS中10分钟；然后将该探针置于含100nM的抗体中，固化高度在1nm，进一步将探针与100nM抗原进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数，获得多个高亲和力嵌合抗体分子(图5-9；表2-6)。The binding kinetic parameters of chimeric antibodies and antigen human FRα (nearshore protein #C784) were analyzed using the Fortebio (BLITZ pro1.1.0.28) instrument. Before the measurement, the AHC biological probe (AHC, Satorius #18-5064) was soaked in PBS for 10 minutes; then the probe was placed in 100nM antibody, with a solidification height of 1nm, and the probe was further combined with 100nM antigen for 400 seconds; then the probe was transferred to PBS for a dissociation reaction for 600 seconds. After the experiment was completed, the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed using the software to calculate the kinetic constants of antigen-antibody binding, and multiple high-affinity chimeric antibody molecules were obtained (Figure 5-9; Table 2-6).
表2抗FRα嵌合抗体体外结合能力
Table 2 In vitro binding ability of anti-FRα chimeric antibodies
表3抗FRα嵌合抗体体外结合能力
Table 3 In vitro binding ability of anti-FRα chimeric antibodies
表4抗FRα嵌合抗体体外结合能力
Table 4 In vitro binding ability of anti-FRα chimeric antibodies
表5抗FRα嵌合抗体体外结合能力
Table 5 In vitro binding ability of anti-FRα chimeric antibodies
表6抗FRα嵌合抗体体外结合能力
Table 6 In vitro binding ability of anti-FRα chimeric antibodies
2.8 FRα嵌合抗体的体外亲和力的流式细胞术评估2.8 Flow cytometric assessment of in vitro affinity of FRα chimeric antibodies
利用细胞膜表面表达FRα的人非小细胞肺癌细胞NCI-H2110(南京科佰#CBP60071)，人卵巢癌细胞SKOV3(南京科佰#CBP60291)等评估候选抗体的结合情况，每个待测抗体样品使用2×105个细胞，使用PBS清洗细胞两次。FRα待测抗体使用1％BSA稀释至高(0.4ug/mL)、中(0.1ug/mL)、低(0.01ug/mL)3个梯度点，使用稀释好的抗体重悬细胞；4℃孵育1小时后，PBS清洗；FITC标记的抗人抗体的二抗(Jackson#109-095-098)稀释1000倍使用(10ul抗体溶液，加入9.99mLPBS稀释为10mL工作液)，100mL/孔重悬细胞，4℃孵育1小时；PBS清洗后流式细胞分析仪(Satorius公司iQue Plus)进行分析(图10-13)。The binding of candidate antibodies was evaluated using human non-small cell lung cancer cells NCI-H2110 (Nanjing Kebai #CBP60071) and human ovarian cancer cells SKOV3 (Nanjing Kebai #CBP60291) expressing FRα on the cell membrane surface. 2×10 5 cells were used for each antibody sample to be tested, and the cells were washed twice with PBS. The FRα test antibody was diluted with 1% BSA to three gradient points: high (0.4ug/mL), medium (0.1ug/mL), and low (0.01ug/mL), and the cells were resuspended with the diluted antibody; incubated at 4°C for 1 hour and washed with PBS; the FITC-labeled anti-human antibody secondary antibody (Jackson #109-095-098) was diluted 1000 times and used (10ul antibody solution, added with 9.99mL PBS to dilute to 10mL working solution), the cells were resuspended in 1000mL/well, and incubated at 4°C for 1 hour; after washing with PBS, the cells were analyzed by flow cytometer (iQue Plus, Satorius) (Figures 10-13).
从图中可知，本发明的抗体，特别是2F22、Pha3等在不同浓度下均与细胞结合，且显示与对照抗体MIRV(Mirvecuximab)相当的结合能力。As can be seen from the figure, the antibodies of the present invention, especially 2F22, Pha3, etc., bind to cells at different concentrations and show binding abilities comparable to those of the control antibody MIRV (Mirvecuximab).
2.9 FRα嵌合抗体的体外细胞内吞实验评估2.9 In vitro endocytosis evaluation of FRα chimeric antibodies
FRα嵌合抗体的体外细胞内吞实验评估使用人非小细胞肺癌细胞NCI-H2110作为靶细胞进行实验。对待测抗体使用DOJINDO的LK01试剂盒进行FITC荧光标记，标记后的抗体使用1％BSA稀释至5μg/mL，NCI-H2110细胞培养至状态最佳，每个样品使用2×105个细胞，用PBS清洗细胞两次后重悬至2％BSA中；将细胞和待测抗体按1:1体积混匀，分为三份，分别放置于冰上两份，放置于37℃培养箱一份，孵育4.5小时。孵育完成后，将冰上的一份使用PBS清洗，另一份冰上孵育样本和37℃培养箱一份使用柠檬酸缓冲液，pH2.7缓 冲液浸泡10分钟，将细胞表面未内吞的抗体清除。清洗后PBS重悬，流式仪读取MFI(表6)。The in vitro cellular endocytosis experiment of FRα chimeric antibody was evaluated using human non-small cell lung cancer cells NCI-H2110 as target cells. The antibody to be tested was FITC fluorescently labeled using DOJINDO's LK01 kit. The labeled antibody was diluted to 5μg/mL using 1% BSA. NCI-H2110 cells were cultured to the best state. 2×10 5 cells were used for each sample. The cells were washed twice with PBS and resuspended in 2% BSA; the cells and the antibody to be tested were mixed at a volume of 1:1 and divided into three portions, two of which were placed on ice and one in a 37°C incubator for 4.5 hours. After the incubation, one portion on ice was washed with PBS, and the other sample incubated on ice and one in a 37°C incubator was incubated with citric acid buffer, pH 2.7. Soak in the rinse solution for 10 minutes to remove the unendocytosed antibodies on the cell surface. After washing, resuspend in PBS and read the MFI on a flow cytometer (Table 6).
表7抗体分子诱导FRa+细胞内吞的活性检测
Table 7 Activity detection of antibody molecules inducing FRα+ cell endocytosis
内吞效率计算公式为：内吞效率＝(37℃MFI－4℃MFI)÷4℃MFI(PBS)×100％The formula for calculating the endocytosis efficiency is: endocytosis efficiency = (37°C MFI - 4°C MFI) ÷ 4°C MFI (PBS) × 100%
MIRV.:Mirvetuximab NC:IgG1，阴性对照MIRV.:Mirvetuximab NC:IgG1, negative control
从上表可以看出，接受检测的抗体均表现出诱导内吞活性，其中2F22、Pha3、10L17等，均显示出优于或者相当于MIRV的活性。As can be seen from the above table, all the antibodies tested showed endocytosis-inducing activity, among which 2F22, Pha3, 10L17, etc., all showed activities superior to or equivalent to MIRV.
2.10 FRα嵌合抗体ADC的体外细胞杀伤实验评估2.10 Evaluation of in vitro cell killing assay of FRα chimeric antibody ADC
FRα嵌合抗体ADC的体外细胞杀伤实验评估使用HEK293-hFOLR1(HEK293-FRα)细胞和KB细胞(ATCC#CCL-17)进行。In vitro cell killing assays of FRα chimeric antibody ADCs were performed using HEK293-hFOLR1 (HEK293-FRα) cells and KB cells (ATCC #CCL-17).
ADC如下制备：ADC was prepared as follows:
在pH为7.4的PBS中，2.0-2.6当量TECP还原抗体2小时后，6eq vcMMAE DMA溶液加入到还原后的抗体溶液中，2-8℃搅拌1小时后超滤换液除去DMA和小分子残留。紫外分光光度计测定偶联物在248nm～280nm处的吸光值，计算得到偶联物的浓度。样品分装到冻存管中-80℃保存。样品的DAR值由HPLC-HIC测定。In PBS at pH 7.4, 2.0-2.6 equivalents of TECP were used to reduce the antibody for 2 hours, and then 6eq vcMMAE DMA solution was added to the reduced antibody solution. After stirring at 2-8°C for 1 hour, the solution was ultrafiltered to remove DMA and small molecule residues. The absorbance of the conjugate at 248nm-280nm was measured by UV spectrophotometer, and the concentration of the conjugate was calculated. The samples were dispensed into cryovials and stored at -80°C. The DAR value of the sample was determined by HPLC-HIC.
将细胞密度调整至5×104/mL，100μL/孔铺于96孔白板中。实验第二天，使用培养基(KB细胞培养基为EMEM+10％FBS；293细胞培养基为DMEM+10％FBS)稀释待测抗体至10μg/mL，然后3倍稀释，设置10个梯度。按50μL/孔加入细胞培养板中，二氧化碳培养箱孵育96小时。孵育完毕后，按照100μL/孔加入配置好的Cell titer Glo(promega货号G7570)，裂解10分钟后，酶标仪读取荧光值。数据采用softmax pro7软件进行4参数拟合曲线(图14-21)。Adjust the cell density to 5×10 4 /mL, and plate 100μL/well in a 96-well white plate. On the second day of the experiment, use culture medium (KB cell culture medium is EMEM+10% FBS; 293 cell culture medium is DMEM+10% FBS) to dilute the test antibody to 10μg/mL, then dilute 3 times, and set 10 gradients. Add 50μL/well to the cell culture plate and incubate in a carbon dioxide incubator for 96 hours. After incubation, add the configured Cell titer Glo (promega product number G7570) at 100μL/well, lyse for 10 minutes, and read the fluorescence value with an enzyme reader. The data was fitted with a 4-parameter curve using softmax pro7 software (Figures 14-21).
上述抗体制备ADC后，显示出了对2种FRa+肿瘤细胞的杀伤活性，其中7G23、10L17、13M15在不同的细胞中显示出了优于或者相当于对标抗体ADC的细胞杀伤能力。After the above antibodies were prepared into ADC, they showed killing activity against two types of FRa+ tumor cells, among which 7G23, 10L17, and 13M15 showed cell killing ability that was better than or equivalent to that of the benchmark antibody ADC in different cells.
实施例3鼠源抗FRα抗体人源化及其表征Example 3 Humanization and characterization of mouse anti-FRα antibody
3.1鼠源抗人FRα抗体人源化3.1 Humanization of mouse anti-human FRα antibody
选择高亲和力，高阻断能力的分子，综合Kabat、Chothia的抗体编码方案，确定鼠源抗体的重链和轻链的6个抗原互补决定簇(CDR)的氨基酸序列区域及支撑抗体保守三维构象的框架区域(framework region)。随后通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相 似接近的人源抗体重链可变区序列，选择其抗体框架区序列作为模板，将鼠源抗体重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列，并将框架区个别氨基酸从人源的改回鼠源的。确定回复突变位点，一是对照设计好的人源化抗体序列和原始的鼠源抗体序列，检查有哪些氨基酸的不同；二是检查这些氨基酸是否对支持抗体结构起重要作用或者对与抗原的结合起重要作用，同时需要检查是否有一些潜在的翻译后修饰位点，如N(天冬酰胺)糖基化位点、N脱酰胺化位点、D(天冬氨酸)异构化位点(例如，将Pha3的轻链CDR2上的DG突变为EG，从而消除抗体的分子异构化的潜在风险)等。获得2F22、Pha3、45B1和45A3的人源化抗体。We selected molecules with high affinity and high blocking ability, and combined the antibody coding schemes of Kabat and Chothia to determine the amino acid sequence regions of the six antigen complementary determinants (CDRs) of the heavy and light chains of the mouse antibody and the framework region that supports the conservative three-dimensional conformation of the antibody. We then analyzed and searched for known human antibody sequences and selected the most similar to the mouse antibody. The humanized antibody heavy chain variable region sequence is similar to that of the humanized antibody. The antibody framework region sequence is selected as a template, and the mouse antibody heavy chain CDR is combined with the human antibody framework region to finally generate the humanized antibody heavy chain variable region sequence. The same process is used to generate the humanized antibody light chain variable region sequence, and the individual amino acids in the framework region are changed from human to mouse. To determine the back mutation site, one is to check which amino acids are different by comparing the designed humanized antibody sequence with the original mouse antibody sequence; the second is to check whether these amino acids play an important role in supporting the antibody structure or in binding to the antigen. At the same time, it is necessary to check whether there are some potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization site (for example, mutating DG on the light chain CDR2 of Pha3 to EG, thereby eliminating the potential risk of molecular isomerization of the antibody), etc. Humanized antibodies of 2F22, Pha3, 45B1 and 45A3 are obtained.
将工程改造后人源化抗体可变区重链基因构建到含人单克隆抗体IgG1亚类的重链恒定区基因(SEQ ID NO:73)的哺乳动物细胞表达载体pcDNATM3.1(+)(invitrogen#V790-20)中；轻链可变区基因构建到含人单克隆抗体κ亚类的轻链恒定区基因(SEQ ID NO:87)的哺乳动物细胞表达载体中。构建好的抗人FRα人源化抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用ProteinA纯化得到抗人FRα人源化抗体蛋白。The engineered humanized antibody variable region heavy chain gene was constructed into a mammalian cell expression vector pcDNA TM 3.1(+)(invitrogen#V790-20) containing a heavy chain constant region gene of the human monoclonal antibody IgG1 subclass (SEQ ID NO: 73); the light chain variable region gene was constructed into a mammalian cell expression vector containing a light chain constant region gene of the human monoclonal antibody κ subclass (SEQ ID NO: 87). The constructed anti-human FRα humanized antibody heavy chain vector and light chain vector were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Protein A to obtain the anti-human FRα humanized antibody protein.
人源化抗体序列参见下表8：The humanized antibody sequences are shown in Table 8 below:
表8：人源化抗体的VH、VL和CDR序列。
Table 8: VH, VL and CDR sequences of humanized antibodies.
3.2抗FRα人源化抗体的Octet评估3.2 Octet evaluation of anti-FRα humanized antibodies
利用Fortebio(BLITZ pro1.1.0.28)仪器分析人源化抗体与抗原人FRα(hFOLR1，近岸蛋白#C784)的结合动力学参数。测定前先将AHC生物探针浸泡于PBS中10分钟；然后将该 探针置于含100nM的抗体中，固化高度在1nm，进一步将探针与100nM抗原进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数(图22,23；表9-10)。The binding kinetic parameters of humanized antibody and antigen human FRα (hFOLR1, nearshore protein #C784) were analyzed using Fortebio (BLITZ pro1.1.0.28). Before the measurement, the AHC bioprobe was soaked in PBS for 10 minutes; The probe was placed in 100nM antibody, with a solidification height of 1nm, and the probe was further combined with 100nM antigen for 400 seconds; then the probe was transferred to PBS for dissociation for 600 seconds. After the experiment was completed, the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed using the software to calculate the kinetic constants of antigen-antibody binding (Figures 22, 23; Tables 9-10).
表9：一组抗体和FRα分子之间的亲和力
Table 9: Affinity between a group of antibodies and FRα molecules
表10一组抗体和FRα分子之间的亲和力
Table 10 Affinity between a group of antibodies and FRα molecules
3.3抗FRα人源化抗体的Biacore评估3.3 Biacore evaluation of anti-FRα humanized antibody
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。参考GE公司操作说明，首先在传感芯片CM5分析通道和对照样品通道都偶联抗人Fc抗体(GE公司Human Antibody Capture Kit.Cat#BR-1008-39)，捕获人FRα抗体样品(100nM)，然后在分析通道和对照样品通道一起流过梯度稀释的人FRα抗原(Human FOLR1,近岸蛋白#C784)或恒河猴FRα(NCBI：NP_001181576.1)(起始浓度100nM，1:2梯度稀释)，测定抗体抗原结合后发生的光反应值。经仪器软件拟合分析(5个梯度稀释浓度点)，最终得到抗体的结合常数Kon和解离常数Koff，以及亲和力常数KD。结果参见图24和图25和表11-12。The antibody-antigen interaction force was determined using the GE BIAcore instrument S200. Referring to the GE operating instructions, firstly, the anti-human Fc antibody (GE Human Antibody Capture Kit. Cat#BR-1008-39) was coupled to the sensor chip CM5 analysis channel and the control sample channel to capture the human FRα antibody sample (100nM), and then the gradient dilution of human FRα antigen (Human FOLR1, near shore protein #C784) or rhesus monkey FRα (NCBI: NP_001181576.1) (starting concentration 100nM, 1:2 gradient dilution) was passed through the analysis channel and the control sample channel together to determine the light reaction value after the antibody-antigen binding. After fitting analysis by the instrument software (5 gradient dilution concentration points), the binding constant Kon and dissociation constant Koff of the antibody, as well as the affinity constant KD, were finally obtained. The results are shown in Figures 24 and 25 and Tables 11-12.
表11：抗体与人FRα分子之间的亲和力

Table 11: Affinity between antibodies and human FRα molecules

表12：抗体和cynoFRα的亲和力检测
Table 12: Affinity test of antibodies and cynoFRα
如上可见，hz45A3和hz45B1分子与人和猴FRa的亲和力高于对照分子(MIRV,Mirvetuximab)。As can be seen above, the affinity of hz45A3 and hz45B1 molecules to human and monkey FRa is higher than that of the control molecule (MIRV, Mirvetuximab).
3.4.FRα人源化抗体的体外亲和力的流式评估3.4. Flow Cytometric Assessment of In Vitro Affinity of Humanized FRα Antibodies
使用过表达FRα的HEK293细胞HEK293-hFOLR1，口腔上皮癌细胞KB(ATCC#CCL-17)评估人源化抗体的结合情况，每个样品使用2×105个细胞，使用PBS清洗细胞两次。FRα待测抗体使用1％BSA稀释至高(10μg/mL)、中(1μg/mL)、低(0.1μg/mL)3个梯度点，使用稀释好的抗体重悬细胞；4℃孵育1小时后，PBS清洗；FITC标记的抗人抗体的二抗(Jackson#109-095-098)稀释1000倍使用，100mL/孔重悬细胞，4℃孵育1小时；PBS清洗后流式细胞分析仪进行分析(图26,27)。HEK293 cells overexpressing FRα, HEK293-hFOLR1, and oral epithelial cancer cells KB (ATCC#CCL-17) were used to evaluate the binding of humanized antibodies. 2×10 5 cells were used for each sample and the cells were washed twice with PBS. The FRα test antibody was diluted with 1% BSA to three gradient points: high (10μg/mL), medium (1μg/mL), and low (0.1μg/mL). The cells were resuspended with the diluted antibody; incubated at 4℃ for 1 hour and washed with PBS; the FITC-labeled anti-human antibody secondary antibody (Jackson#109-095-098) was diluted 1000 times and the cells were resuspended in 100mL/well and incubated at 4℃ for 1 hour; after washing with PBS, the cells were analyzed by flow cytometer (Figures 26, 27).
可见，工程化改造过的分子表现出了不同的亲和力，其中2F22-H1L0、Pha3-H1L2等分子显示出了优于或者相当对标分子的FRa+细胞结合能力。It can be seen that the engineered molecules showed different affinities, among which molecules such as 2F22-H1L0 and Pha3-H1L2 showed FRa+ cell binding ability that was better than or equivalent to the benchmark molecules.
3.5.FRα人源化抗体的体外亲和力的流式评估3.5. Flow Cytometric Assessment of In Vitro Affinity of Humanized FRα Antibodies
使用过表达FRα的HEK293细胞HEK293-hFOLR1(HEK293-hFRα)_，以及口腔上皮癌细胞KB(ATCC#CCL-17)评估人源化抗体的结合情况，每个样品使用2×105个细胞，使用PBS清洗细胞两次。FRα待测抗体使用含1％BSA的FBS溶液做1:4梯度稀释，将200ul抗体溶液，起始浓度为10ug/mL，依次加入到含有400uL FBS_1％BSA溶液的下一个稀释度试管，共得到7个稀释浓度。使用稀释好的抗体重悬细胞至100uL，4℃孵育1小时后PBS清洗；使用FBS_1％BSA稀释1000倍的FITC标记的抗人抗体的二抗(Jackson#109-095-098)，100uL/孔重悬细胞，4℃孵育1小时后PBS清洗。使用流式细胞分析仪进行分析(图28和29和表13、14)。HEK293 cells overexpressing FRα, HEK293-hFOLR1 (HEK293-hFRα)_, and oral epithelial cancer cells KB (ATCC#CCL-17) were used to evaluate the binding of humanized antibodies. 2×10 5 cells were used for each sample and the cells were washed twice with PBS. The FRα test antibody was diluted 1:4 with FBS solution containing 1% BSA. 200ul of antibody solution, with a starting concentration of 10ug/mL, was added to the next dilution tube containing 400uL FBS_1% BSA solution in sequence, resulting in a total of 7 dilution concentrations. The cells were resuspended to 100uL with the diluted antibody, incubated at 4℃ for 1 hour, and then washed with PBS; the secondary antibody (Jackson#109-095-098) of FITC-labeled anti-human antibody diluted 1000 times with FBS_1% BSA was used, and the cells were resuspended at 100uL/well, incubated at 4℃ for 1 hour, and then washed with PBS. The analysis was performed using a flow cytometer ( FIGS. 28 and 29 and Tables 13 and 14 ).
表13：抗体同过表达FRα的HEK293细胞的结合能力
Table 13: Binding ability of antibodies to HEK293 cells overexpressing FRα
表14：抗体同过表达FRα的KB细胞的结合能力
Table 14: Binding ability of antibodies to KB cells overexpressing FRα
可见，hz45A3、hz45B1等分子显示出了优于对照分子(Mirv,Mirvetuximab)结合细胞 表面FRa的能力。It can be seen that hz45A3, hz45B1 and other molecules showed better binding to cells than control molecules (Mirv, Mirvetuximab) The ability of surface FRa.
3.6FRα人源化抗体的体外细胞内吞实验评估In vitro endocytosis evaluation of 3.6FRα humanized antibody
使用人非小细胞肺癌细胞NCI-H2110作为靶细胞，进行FRα人源化抗体的体外细胞内吞能力评估。对待测抗体使用DOJINDO的LK01试剂盒进行FITC荧光标记，标记后的抗体用1％BSA稀释至5μg/mL，每个样品使用2E5个细胞，用PBS清洗细胞后重悬至2％BSA中；将细胞和待测抗体按1:1体积混匀后分为三份，放置于冰上两份，37℃培养箱一份，孵育4.5小时。孵育完成后，将冰上的一份使用PBS清洗，另一份和37℃培养箱一份使用酸洗10分钟，将细胞表面未内吞的抗体清除。清洗后PBS重悬流式仪读取MFI(表15)。Human non-small cell lung cancer cells NCI-H2110 were used as target cells to evaluate the in vitro cellular endocytosis ability of FRα humanized antibodies. The antibody to be tested was FITC fluorescently labeled using DOJINDO's LK01 kit. The labeled antibody was diluted to 5 μg/mL with 1% BSA. 2E5 cells were used for each sample. The cells were washed with PBS and resuspended in 2% BSA; the cells and the antibody to be tested were mixed at a volume of 1:1 and divided into three parts, two of which were placed on ice and one in a 37°C incubator for 4.5 hours. After incubation, one part on ice was washed with PBS, and the other part and one part in a 37°C incubator were acid-washed for 10 minutes to remove the antibodies that were not internalized on the cell surface. After washing, the PBS was resuspended in the flow cytometer to read the MFI (Table 15).
内吞效率计算公式为：内吞效率＝(37℃MFI－4℃MFI)÷4℃MFI(PBS)×100％The formula for calculating the endocytosis efficiency is: endocytosis efficiency = (37°C MFI - 4°C MFI) ÷ 4°C MFI (PBS) × 100%
可见，在FRα+的细胞NCI-H2110中，工程化改造过的抗体分子表现出了诱导FRa+细胞内吞能力，其中2F22-H1L0、2F22-H1L1、Phage3-H1L2、Hz45A3和Hz45B1等分子都显示出了优于对照分子(MIRV，Mirvetuximab)的FRa+细胞内吞能力。It can be seen that in the FRα+ cell NCI-H2110, the engineered antibody molecules showed the ability to induce FRa+ cell endocytosis, among which 2F22-H1L0, 2F22-H1L1, Phage3-H1L2, Hz45A3 and Hz45B1 molecules all showed FRa+ cell endocytosis ability better than the control molecule (MIRV, Mirvetuximab).
表15：抗体分子诱导FRa+细胞内吞的活性检测
Table 15: Activity detection of antibody molecules inducing FRα+ cell endocytosis
3.7 FRα人源化抗体ADC的体外细胞杀伤实验评估3.7 In vitro cell killing assay evaluation of FRα humanized antibody ADC
人源化抗体ADC的制备：Preparation of humanized antibody ADC:
在pH为7.4的PBS中，2.0-2.6当量TECP还原抗体2小时后，6eq vcMMAE DMA溶液加入到还原后的抗体溶液中，2-8℃搅拌1小时后超滤换液除去DMA和小分子残留。制备获得Pha-H0L0-ADC(也表示为hzPha-H0L0-MMAE)、Pha-H0L1-ADC(也表示为hzPha-H0L1-MMAE)、Pha-H1L2-ADC(也表示为hzPha-H1L2-MMAE)、2F22-H1L0-ADC(也表示为2F22-H1L0-MMAE)、2F22-H1L1-ADC(也表示为2F22-H1L1-MMAE)、hz45A3-ADC(也表示为hz45A3-MMAE)、hz45B1-ADC(也表示为hz45B1-MMAE)、MIRV-ADC(也表示为MIRV-MMAE或Mirve-MMAE)和NC-ADC(IgG1-MMAE)。紫外分光光度计测定偶联物在248nm～280nm处的吸光值，计算得到偶联物的浓度。样品分装到冻存管中-80℃保存。样品的DAR值由HPLC-HIC测定。In PBS at pH 7.4, 2.0-2.6 equivalents of TECP were used to reduce the antibody for 2 hours, and then 6eq vcMMAE DMA solution was added to the reduced antibody solution. After stirring at 2-8°C for 1 hour, the solution was ultrafiltered to remove DMA and small molecule residues. Pha-H0L0-ADC (also expressed as hzPha-H0L0-MMAE), Pha-H0L1-ADC (also expressed as hzPha-H0L1-MMAE), Pha-H1L2-ADC (also expressed as hzPha-H1L2-MMAE), 2F22-H1L0-ADC (also expressed as 2F22-H1L0-MMAE), 2F22-H1L1-ADC (also expressed as 2F22-H1L1-MMAE), hz45A3-ADC (also expressed as hz45A3-MMAE), hz45B1-ADC (also expressed as hz45B1-MMAE), MIRV-ADC (also expressed as MIRV-MMAE or Mirve-MMAE) and NC-ADC (IgG1-MMAE) were prepared. The absorbance of the conjugate at 248nm to 280nm was measured by ultraviolet spectrophotometer, and the concentration of the conjugate was calculated. The samples were divided into cryovials and stored at -80°C. The DAR values of the samples were determined by HPLC-HIC.
FRα人源化抗体ADC的体外细胞杀伤评估使用HEK293-FRα细胞和KB细胞(ATCC#CCL-17)进行。HEK293-FRα细胞和KB细胞密度分别调整至5×104/mL，100μL/孔铺于96孔白板中。实验第二天，使用培养基稀释待测抗体ADC分子至10μg/mL，然后3 倍稀释，设置10个梯度。按50μL/孔加入细胞培养板中，二氧化碳培养箱孵育96小时。孵育完毕后，按照100μL/孔加入配置好的Cell titer Glo(promega货号G7570)，裂解10分钟后，酶标仪读取荧光值。数据采用softmax pro7软件进行4参数拟合曲线(图30-34：HEK293-FRα(HEK293-hFOLR1)细胞，图35：KB细胞)。In vitro cell killing evaluation of FRα humanized antibody ADC was performed using HEK293-FRα cells and KB cells (ATCC#CCL-17). The density of HEK293-FRα cells and KB cells was adjusted to 5×10 4 /mL, and 100 μL/well was plated in a 96-well white plate. On the second day of the experiment, the antibody ADC molecule to be tested was diluted to 10 μg/mL using culture medium, and then Dilution was performed by 10 times, and 10 gradients were set. 50 μL/well was added to the cell culture plate and incubated in a carbon dioxide incubator for 96 hours. After incubation, 100 μL/well of the configured Cell titer Glo (Promega catalog number G7570) was added. After lysis for 10 minutes, the fluorescence value was read by a microplate reader. The data were fitted with a 4-parameter curve using softmax pro7 software (Figures 30-34: HEK293-FRα (HEK293-hFOLR1) cells, Figure 35: KB cells).
可见，工程化改造过的分子制备ADC，表现出了FRa+细胞杀伤能力，其中2F22-H1L1、Pha3-H1L2、Hz45A3和Hz45B1等的ADC分子显示出了和对照分子(MIRV Mirvetuximab)的ADC相当的FRa+细胞杀伤能力。It can be seen that the ADCs prepared by engineered molecules exhibited FRa+ cell killing ability, among which ADC molecules such as 2F22-H1L1, Pha3-H1L2, Hz45A3 and Hz45B1 showed FRa+ cell killing ability comparable to that of the control molecule (MIRV Mirvetuximab).
3.8 FRα人源化抗体大鼠体内药代动力学研究3.8 Pharmacokinetic study of FRα humanized antibody in rats
用0.9％氯化钠注射液稀释待测抗体(hzPha3H1L2、hz45B1、hz45A3、MIRV)至2mg/mL浓度，每个抗体使用5只雄性SD大鼠，按照10mg/Kg计算所需体积，尾静脉单次给药后，在1min、2h、8h、D2(24h)、D3(48h)、D5(96h)、D6(120h)、D8(168h)、D11(240h)、D15(336h)各时间点，颈静脉采血约0.2mL并分离血清；采用常规ELISA方法检测样本中受试抗体浓度，评价不同抗体分子在大鼠体内的药代动力学。结果见图36和表16。The antibodies to be tested (hzPha3H1L2, hz45B1, hz45A3, MIRV) were diluted to a concentration of 2 mg/mL with 0.9% sodium chloride injection. Five male SD rats were used for each antibody, and the required volume was calculated according to 10 mg/Kg. After a single dose of tail vein administration, about 0.2 mL of blood was collected from the jugular vein at each time point of 1 min, 2 h, 8 h, D2 (24 h), D3 (48 h), D5 (96 h), D6 (120 h), D8 (168 h), D11 (240 h), and D15 (336 h) and serum was separated; the concentration of the tested antibodies in the samples was detected by conventional ELISA method to evaluate the pharmacokinetics of different antibody molecules in rats. The results are shown in Figure 36 and Table 16.
表16：hzPha3H1L2、hz45B1、hz45A3、MIRV大鼠体内的药代动力学参数
Table 16: Pharmacokinetic parameters of hzPha3H1L2, hz45B1, hz45A3, and MIRV in rats
可见，hzPha3H1L2、hz45B1、hz45A3分子在大鼠体内同对照分子(MIRV，Mirvetuximab)有相近的药代动力学特性。It can be seen that hzPha3H1L2, hz45B1, and hz45A3 molecules have similar pharmacokinetic properties to the control molecule (MIRV, Mirvetuximab) in rats.
3.9 FRα人源化抗体小鼠体内药效研究3.9 In vivo efficacy study of FRα humanized antibody in mice
委托中国科学院上海药物研究所评价并比较hzPha3-H1L2-MMAE、hz45B1-MMAE、hz45A3-MMAE、Mirve-MMAE对人宫颈癌KB裸小鼠皮下移植瘤的疗效。The Shanghai Institute of Materia Medica, Chinese Academy of Sciences was commissioned to evaluate and compare the efficacy of hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, and Mirve-MMAE on subcutaneous transplanted tumors of human cervical cancer KB nude mice.
使用4周龄雌性BALB/c nu/nu裸小鼠，皮下接种KB细胞，待肿瘤长到100～150mm3后，根据肿瘤体积分为8组，将受试药物(hzPha3-H1L2-MMAE、hz45B1-MMAE、hz45A3-MMAE、Mirve-MMAE)用PBS配置为0.1mg/mL和0.3mg/mL，静脉注射(IV)药物，注射体积0.1mL/10g体重。分组和给药情况见表17。每周二次用游标卡尺测量肿瘤直径，肿瘤体积(V)，计算公式为Four-week-old female BALB/c nu/nu nude mice were subcutaneously inoculated with KB cells. After the tumor grew to 100-150 mm3 , they were divided into 8 groups according to the tumor volume. The test drugs (hzPha3-H1L2-MMAE, hz45B1-MMAE, hz45A3-MMAE, Mirve-MMAE) were prepared with PBS at 0.1 mg/mL and 0.3 mg/mL, and intravenously (IV) injected with an injection volume of 0.1 mL/10 g body weight. The grouping and dosing are shown in Table 17. The tumor diameter was measured with a vernier caliper twice a week, and the tumor volume (V) was calculated as follows:
V＝1/2×a×b2，其中a、b分别表示长、宽。V = 1/2 × a × b 2 , where a and b represent length and width respectively.
评价并比较不同分子的ADC对人宫颈癌KB裸小鼠皮下移植瘤的疗效。评价指标为T/C％或肿瘤生长抑制率(TGI％)：Evaluate and compare the efficacy of ADCs with different molecules on subcutaneous transplanted tumors of human cervical cancer KB nude mice. The evaluation index is T/C% or tumor growth inhibition rate (TGI%):
T/C(％)-(T-T0)/(C-C0)×100，其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积，C代表对照，T代表实验组肿瘤体积。T/C (%) - (TT 0 ) / (CC 0 ) × 100, where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment, C represents the control group, and T represents the tumor volume of the experimental group.
肿瘤生长抑制率％：(TGI％)＝100-T/C(％)。 Tumor growth inhibition rate %: (TGI %) = 100-T/C (%).
当肿瘤出现消退时，肿瘤生长抑制率％：(TGI％)＝100-(T-T0)/T0×100When the tumor regresses, the tumor growth inhibition rate (%) is: (TGI%) = 100-(TT 0 )/T 0 × 100
如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。If the tumor is smaller than its initial volume, that is, T<T 0 or C<C 0 , it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
实验结束，CO2麻醉处死动物，随后解剖取瘤并拍照。At the end of the experiment, the animals were killed by CO2 anesthesia, and then the tumors were dissected and photographed.
结果见图37、38和表17。
The results are shown in Figures 37, 38 and Table 17.
如图37、38和表17所示，hzPha3-H1L2-MMAE(1、3mg/kg，IV，D0)剂量依赖性地显著抑制KB裸小鼠皮下移植瘤的生长，抑瘤率分别为64％和98％，3mg/kg剂量组有5/6小鼠肿瘤部分消退；hz45B1-MMAE(1、3mg/kg，IV，D0)对KB裸小鼠皮下移植瘤的抑瘤率分别为59％和128％，3mg/kg剂量组有6/6小鼠肿瘤部分消退；hz45A3-MMAE(1、3mg/kg，IV，D0)对KB裸小鼠皮下移植瘤的抑瘤率分别为58％和101％，3mg/kg剂量组有5/6小鼠肿瘤部分消退；Mirve-MMAE(1、3mg/kg，IV，D0)对KB裸小鼠皮下移植瘤的抑瘤率分别为69％和99％，3mg/kg剂量组有3/6小鼠肿瘤部分消退。As shown in Figures 37, 38 and Table 17, hzPha3-H1L2-MMAE (1, 3 mg/kg, IV, D0) significantly inhibited the growth of subcutaneous transplanted tumors in KB nude mice in a dose-dependent manner, with tumor inhibition rates of 64% and 98%, respectively. In the 3 mg/kg dose group, 5/6 mice had partial tumor regression; hz45B1-MMAE (1, 3 mg/kg, IV, D0) had tumor inhibition rates of 59% and 128%, respectively. The tumors of 6/6 mice in the dosage group partially regressed; the tumor inhibition rates of hz45A3-MMAE (1, 3 mg/kg, IV, D0) on subcutaneous transplanted tumors in KB nude mice were 58% and 101%, respectively, and the tumors of 5/6 mice in the 3 mg/kg dosage group partially regressed; the tumor inhibition rates of Mirve-MMAE (1, 3 mg/kg, IV, D0) on subcutaneous transplanted tumors in KB nude mice were 69% and 99%, respectively, and the tumors of 3/6 mice in the 3 mg/kg dosage group partially regressed.
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Claims (40)

  1. 结合FRα的抗体或其抗原结合片段，所述抗体包含An antibody or antigen-binding fragment thereof that binds to FRα, the antibody comprising
    1)如SEQ ID NO:116、34或115所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；1) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 116, 34 or 115, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 51;
    2)如SEQ ID NO:116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；2) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:116, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:51;
    3)如SEQ ID NO:115或34所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；3) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 115 or 34, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 51;
    4)如SEQ ID NO:1或13所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:2、24或25所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；4) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1 or 13, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, 24 or 25;
    5)如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:2所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；5) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2;
    6)如SEQ ID NO:13所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:24或25所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；6) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:13, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:24 or 25;
    7)如SEQ ID NO:41、33或34所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42、39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；7) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:41, 33 or 34, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:42, 39, 40 or 51;
    8)如SEQ ID NO:33所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；8) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:33, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39, 40 or 51;
    9)如SEQ ID NO:34所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；9) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:34, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39, 40 or 51;
    10)如SEQ ID NO:115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；10) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 115 or 116, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 39, 40 or 51;
    11)如SEQ ID NO:115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；11) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 115 or 116, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 51;
    12)如SEQ ID NO:41所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3； 12) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:41, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:42;
    13)如SEQ ID NO:74、99、115或116所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:42、39、40或51所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；13) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 74, 99, 115 or 116, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 42, 39, 40 or 51;
    14)如SEQ ID NO:14所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:15所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；14) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15;
    15)如SEQ ID NO:26所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:27所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；15) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:26, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27;
    16)如SEQ ID NO:35所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:36所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；16) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:35, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:36;
    17)如SEQ ID NO:53所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:54所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；17) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:53, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:54;
    18)如SEQ ID NO:65所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:66所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；18) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:65, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:66;
    19)如SEQ ID NO:75所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:76所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；19) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:76;
    20)如SEQ ID NO:88所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:89所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；20) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:88, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:89;
    21)如SEQ ID NO:100所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:101所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；21) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 100, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 101;
    22)如SEQ ID NO:109所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:110所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；22) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 109, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 110;
    23)如SEQ ID NO:117所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO:118所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；23) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 117, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 118;
    24)如SEQ ID NO:74或99所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：42所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；24) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 74 or 99, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 42;
    优选地，其中所述HCDR和所述LCDR根据Chothia或Kabat确定，或根据Chothia和Kabat确定； Preferably, wherein said HCDR and said LCDR are determined according to Chothia or Kabat, or according to Chothia and Kabat;
    例如，所述HCDR根据Kabat方案确定，所述LCDR根据Kabat和Chothia组合方案(Kabat&Chothia)确定；For example, the HCDR is determined according to the Kabat scheme, and the LCDR is determined according to the Kabat and Chothia combined scheme (Kabat &Chothia);
    例如，所述HCDR根据Chothia方案确定，所述LCDR根据Kabat和Chothia组合方案(Kabat&Chothia)确定。For example, the HCDR is determined according to the Chothia scheme, and the LCDR is determined according to the combined Kabat and Chothia scheme (Kabat & Chothia).
  2. 结合FRα的抗体或其抗原结合片段，所述抗体包含重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2和HCDR3基于Chothia方案确定，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成，且所述LCDR1、LCDR2和LCDR3基于Kabat和Chothia组合方案，其分别包含由下表SEQ ID NO所示的氨基酸序列组成：
    An antibody or antigen-binding fragment thereof that binds to FRα, the antibody comprising three complementary determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and three complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region, wherein the HCDR1, HCDR2 and HCDR3 are determined based on the Chothia scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NO, and the LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combination scheme, and they respectively comprise and consist of the amino acid sequences shown in the following table SEQ ID NO:
    或者其中所述HCDR1、HCDR2和HCDR3基于Kabat方案确定，其分别包含或由下表SEQ ID NO所示的氨基酸序列组成，且所述LCDR1、LCDR2和LCDR3基于Kabat和Chothia组合方案，其分别包含由下表SEQ ID NO所示的氨基酸序列组成：

    Or wherein the HCDR1, HCDR2 and HCDR3 are determined based on the Kabat scheme, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs, and the LCDR1, LCDR2 and LCDR3 are based on the Kabat and Chothia combined schemes, and they respectively comprise or consist of the amino acid sequences shown in the following table SEQ ID NOs:

  3. 如权利要求1或2所述的结合FRα的抗体或其抗原结合片段，所述抗体包含重链可变区，其中所述重链可变区包含与选自SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116所示的氨基酸序列具有至少90％同一性的氨基酸序列，或由所述序列组成或包含选自SEQ ID NO:1、14、26、35、41、53、65、75、88、100、109、117、13、33、34、74、99、115或116所示的氨基酸序列，或由所述序列组成。An antibody or antigen-binding fragment thereof that binds to FRα as described in claim 1 or 2, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116, or consists of the sequence, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 26, 35, 41, 53, 65, 75, 88, 100, 109, 117, 13, 33, 34, 74, 99, 115 or 116, or consists of the sequence.
  4. 如权利要求1或2所述的结合FRα的抗体或其抗原结合片段，所述抗体包含轻链可变区，其中所述轻链可变区包含与选自SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51所示的氨基酸序列具有至少90％同一性的氨基酸序列，或由所述序列组成或包含选自SEQ ID NO:2、15、27、36、42、54、66、76、89、101、110、118、24、25、39、40或51所示的氨基酸序列，或由所述序列组成。An antibody or antigen-binding fragment thereof that binds to FRα as described in claim 1 or 2, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51, or consists of the sequence, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 27, 36, 42, 54, 66, 76, 89, 101, 110, 118, 24, 25, 39, 40 or 51, or consists of the sequence.
  5. 如权利要求1或2所述的结合FRα的抗体或其抗原结合片段，所述抗体包含重链可变区和轻链可变区，其中所述抗体或其抗原结合片段包含的重链可变区和轻链可变区包含如下表SEQ ID NO所示的氨基酸序列或由所述氨基酸序列组成：

    The antibody or antigen-binding fragment thereof that binds to FRα according to claim 1 or 2, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprised by the antibody or antigen-binding fragment thereof comprise or consist of the amino acid sequence shown in SEQ ID NO in the following table:

  6. 如权利要求1-5中任一项所述的结合FRα的抗体或其抗原结合片段，所述抗体包含重链恒定区，例如IgG1、IgG2、IgG3或IgG4的重链恒定区，例如IgG1的重链恒定区，其The antibody or antigen-binding fragment thereof that binds to FRα according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain constant region, such as a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as a heavy chain constant region of IgG1,
    (i)包含与选自SEQ ID NO:63的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 63;
    (ii)包含选自SEQ ID NO:63的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:63; or
    (iii)包含与选自SEQ ID NO:63的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:63.
  7. 如权利要求1-6中任一项所述的结合FRα的抗体或其抗原结合片段，所述抗体包含轻链恒定区，例如lambda或κ轻链恒定区，例如所述轻链恒定区The antibody or antigen-binding fragment thereof that binds to FRα according to any one of claims 1 to 6, wherein the antibody comprises a light chain constant region, such as a lambda or kappa light chain constant region, such as the light chain constant region
    (i)包含与选自SEQ ID NO:64的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 64;
    (ii)包含选自SEQ ID NO:64的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:64; or
    (iii)包含与选自SEQ ID NO:64的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:64.
  8. 如权利要求1-7中任一项所述的结合FRα的抗体或其抗原结合片段，所述抗体包含 重链恒定区和轻链恒定区，其中The antibody or antigen-binding fragment thereof that binds to FRα according to any one of claims 1 to 7, wherein the antibody comprises The heavy chain constant region and the light chain constant region, wherein
    重链恒定区包含SEQ ID NO:63的氨基酸序列或由其组成；和轻链恒定区包含SEQ ID NO:64的氨基酸序列或由其组成。The heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:63; and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:64.
  9. 权利要求1或2所述的结合FRα的抗体或其抗原结合片段，其具有以下一个或多个特性：The antibody or antigen-binding fragment thereof that binds to FRα according to claim 1 or 2, which has one or more of the following characteristics:
    (1)能够以高亲和力结合人或恒河猴FRα；(1) Ability to bind human or rhesus monkey FRα with high affinity;
    (2)能够以高亲和力结合细胞膜表面表达的人或恒河猴FRα；(2) Ability to bind to human or rhesus monkey FRα expressed on the cell membrane surface with high affinity;
    (3)能够诱导FRα阳性细胞对抗体或其片段或包含所述抗体或其片段的分子的内吞作用，优选地具有相当于已知抗FRα抗体(例如MIRV)或优于已知抗FRα抗体(例如MIRV)的内吞效率；(3) capable of inducing endocytosis of the antibody or fragment thereof or a molecule comprising the antibody or fragment thereof by FRα-positive cells, preferably with an endocytosis efficiency equivalent to or better than that of known anti-FRα antibodies (e.g., MIRV);
    (4)包含其的分子具有细胞(例如肿瘤细胞，例如FRα阳性细胞，例如FRα阳性肿瘤细胞)杀伤能力。(4) The molecule comprising the same has the ability to kill cells (eg, tumor cells, eg, FRα-positive cells, eg, FRα-positive tumor cells).
    (5)显示与权利要求8所列的任一抗体对FRα相同或相似的结合亲和力和/或特异性；(5) showing the same or similar binding affinity and/or specificity for FRα as any of the antibodies listed in claim 8;
    (6)抑制权利要求8所列的任一抗体与FRα的结合；(6) inhibiting the binding of any antibody listed in claim 8 to FRα;
    (7)与权利要求8所示的任一抗体结合相同或重叠的表位；(7) binding to the same or overlapping epitope as any of the antibodies set forth in claim 8;
    (8)与权利要求8所示的任一抗体竞争结合FRα；或(8) competing with any antibody of claim 8 for binding to FRα; or
    (9)具有权利要求8所列的任一抗体分子的一个或多个生物学特性。(9) Having one or more biological properties of any antibody molecule listed in claim 8.
  10. 权利要求9所述的抗体或其抗原结合片段，其中：The antibody or antigen-binding fragment thereof according to claim 9, wherein:
    (1)以高亲和力结合人FRα，优选以下平衡解离常数KD与人FRα结合，所述KD小于5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM或0.08nM。(1) binds to human FRα with high affinity, preferably with an equilibrium dissociation constant, KD , of less than 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM or 0.08 nM.
  11. 权利要求1-10中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是IgG1、IgG2、IgG3或IgG4形式的抗体或其抗原结合片段。The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof in the form of IgG1, IgG2, IgG3 or IgG4.
  12. 权利要求1-11中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是IgG1形式的抗体或其抗原结合片段，并且包含人Kappa轻链恒定区。The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof in the form of IgG1 and comprises a human kappa light chain constant region.
  13. 权利要求1-12中任一项所述的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody is a monoclonal antibody.
  14. 权利要求1-13中任一项所述的抗体或其抗原结合片段，其中所述抗体是人源化的抗体或嵌合抗体。The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a humanized antibody or a chimeric antibody.
  15. 权利要求1-14中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体例如scFv、(Fab’)2片段、单结构域抗体、双抗体或线性抗体。The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv, (Fab') 2 fragments, single domain antibodies, diabodies or linear antibodies.
  16. 权利要求15所述的抗体或其抗原结合片段，其中所述单链抗体是scFv。The antibody or antigen-binding fragment thereof of claim 15, wherein the single-chain antibody is a scFv.
  17. 权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体是双特异性抗体或多特异性抗体。 The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a bispecific antibody or a multispecific antibody.
  18. 分离的核酸，其编码权利要求1至17中任一项的抗FRα抗体或其抗原结合片段。An isolated nucleic acid encoding the anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.
  19. 包含权利要求18的核酸的表达载体。An expression vector comprising the nucleic acid of claim 18.
  20. 权利要求19的表达载体，其中所述表达载体为pcDNA3.1载体。The expression vector of claim 19, wherein the expression vector is a pcDNA3.1 vector.
  21. 包含权利要求18的核酸或权利要求19或20的表达载体的宿主细胞。A host cell comprising the nucleic acid of claim 18 or the expression vector of claim 19 or 20.
  22. 权利要求21所述的宿主细胞，其中所述宿主细胞是原核的或真核的。The host cell of claim 21, wherein the host cell is prokaryotic or eukaryotic.
  23. 权利要求22所述的宿主细胞，其中所述宿主细胞选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。The host cell of claim 22, wherein the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  24. 权利要求23所述的宿主细胞，其中所述宿主细胞是293细胞或CHO细胞。The host cell of claim 23, wherein the host cell is a 293 cell or a CHO cell.
  25. 制备抗FRα抗体或其抗原结合片段的方法，所述方法包括在适于表达编码权利要求1至17中任一项的抗FRα抗体或其抗原结合片段的核酸的条件下培养权利要求21-23中任一项所述的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗FRα抗体或其抗原结合片段。A method for preparing an anti-FRα antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell of any one of claims 21-23 under conditions suitable for expressing a nucleic acid encoding the anti-FRα antibody or antigen-binding fragment thereof of any one of claims 1 to 17, optionally isolating the antibody or antigen-binding fragment thereof, and optionally the method further comprises recovering the anti-FRα antibody or antigen-binding fragment thereof from the host cell.
  26. 免疫缀合物，其包含权利要求1至17中任一项的抗体或其抗原结合片段和其它物质。An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 and other substances.
  27. 权利要求26所述的免疫缀合物，其中所述其它物质是细胞毒性剂。The immunoconjugate of claim 26, wherein the other substance is a cytotoxic agent.
  28. 药物组合物，其包含权利要求1至17中任一项的抗体或其抗原结合片段或者权利要求26或27的免疫缀合物，以及任选地药用辅料。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the immunoconjugate according to claim 26 or 27, and optionally a pharmaceutically acceptable excipient.
  29. 药物组合物，其包含权利要求1至17中任一项的抗体或其抗原结合片段或者权利要求26或27的免疫缀合物，以及其它治疗剂，以及任选地药用辅料。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the immunoconjugate according to claim 26 or 27, and other therapeutic agents, and optionally pharmaceutically acceptable excipients.
  30. 权利要求29所述的药物组合物，其中所述其它治疗剂选自化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂或免疫调节剂。The pharmaceutical composition of claim 29, wherein the other therapeutic agent is selected from a chemotherapeutic agent, other antibodies, cytotoxic agents, vaccines, anti-infective agents or immunomodulators.
  31. 权利要求30所述的药物组合物，其中所述免疫调节剂是共刺激分子的激活剂或免疫检查点分子的抑制剂。The pharmaceutical composition of claim 30, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.
  32. 有效量的权利要求1至17中任一项的抗FRα抗体或其抗原结合片段、或者权利要求26或27的免疫缀合物、或权利要求28至31中任一项的药物组合物在制备药物中的用途，所述药物用于在受试者中预防或治疗受试者或个体肿瘤。Use of an effective amount of an anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or an immunoconjugate according to claim 26 or 27, or a pharmaceutical composition according to any one of claims 28 to 31 in the preparation of a medicament for preventing or treating a tumor in a subject or an individual.
  33. 权利要求32中任一项所述的用途，其中所述肿瘤是实体肿瘤。The use according to any one of claim 32, wherein the tumor is a solid tumor.
  34. 权利要求32或33的用途，其中所述肿瘤是癌症。The use of claim 32 or 33, wherein the tumor is cancer.
  35. 权利要求34所述的用途，其中所述癌症是肺癌(例如非小细胞肺癌)、口腔癌(如口腔上皮癌)、卵巢癌、乳腺癌、间质瘤、或子宫内膜癌。The use of claim 34, wherein the cancer is lung cancer (e.g., non-small cell lung cancer), oral cancer (e.g., oral epithelial cancer), ovarian cancer, breast cancer, stromal tumor, or endometrial cancer.
  36. 权利要求32-35中任一项所述的用途，其还包括向所述受试者联合施用一种或多种其它疗法。The use of any one of claims 32-35, further comprising administering to the subject in combination one or more other therapies.
  37. 权利要求36所述的用途，其中所述疗法包括治疗方式和/或其它治疗剂。The use of claim 36, wherein the therapy comprises a therapeutic modality and/or other therapeutic agents.
  38. 权利要求37所述的用途，其中所述其它治疗剂选自化疗剂、细胞因子、其它抗体、细胞毒性剂、疫苗、小分子药物或免疫调节剂，和/或所述治疗方式包括手术治疗和/或放射 疗法。The use of claim 37, wherein the other therapeutic agent is selected from a chemotherapeutic agent, a cytokine, another antibody, a cytotoxic agent, a vaccine, a small molecule drug or an immunomodulator, and/or the treatment comprises surgery and/or radiation. therapy.
  39. 权利要求38所述的用途，其中所述免疫调节剂是共刺激分子的激活剂或免疫检查点分子的抑制剂。The use of claim 38, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.
  40. 检测样品中FRα的方法，所述方法包括A method for detecting FRα in a sample, the method comprising
    (a)将样品与权利要求1至17中任一项的任何抗FRα抗体或其抗原结合片段接触；和(a) contacting a sample with any anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 1 to 17; and
    (b)检测抗FRα抗体或其抗原结合片段和FRα间的复合物的形成；任选地，抗FRα抗体是被可检测地标记的。 (b) detecting formation of a complex between the anti-FRα antibody or antigen-binding fragment thereof and FRα; optionally, the anti-FRα antibody is detectably labeled.
PCT/CN2023/132065 2022-11-16 2023-11-16 FRα-TARGETING ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, AND USE THEREOF WO2024104431A1 (en)

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