WO2019178852A1 - Ox40抗原多肽及其用途 - Google Patents

Ox40抗原多肽及其用途 Download PDF

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WO2019178852A1
WO2019178852A1 PCT/CN2018/080280 CN2018080280W WO2019178852A1 WO 2019178852 A1 WO2019178852 A1 WO 2019178852A1 CN 2018080280 W CN2018080280 W CN 2018080280W WO 2019178852 A1 WO2019178852 A1 WO 2019178852A1
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amino acid
antigen
antigen polypeptide
isolated
antibody
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PCT/CN2018/080280
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English (en)
French (fr)
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徐霆
彭建建
马树立
栾彦
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苏州丁孚靶点生物技术有限公司
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Priority to PCT/CN2018/080280 priority Critical patent/WO2019178852A1/zh
Priority to CN201880091579.3A priority patent/CN111936507B/zh
Publication of WO2019178852A1 publication Critical patent/WO2019178852A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • the present application relates to the field of antibodies, particularly antigenic polypeptides for the preparation of specific antibodies.
  • the present application relates to isolated OX40 antigen polypeptides, and their use for the production, identification or screening of immunologically active substances, such as antibodies.
  • T cell activation requires two signals to work synergistically: the first signal is produced by the T cell antigen receptor (TCR) recognition antigen, and the signal is transduced into the cell by the CD3 molecule, and the first signal determines the T cell in the adaptive immune response. Specificity; the second signal is produced by interaction of antigen presenting cells (APCs) or co-stimulatory molecules on the surface of target cells with corresponding costimulatory molecule receptors on the surface of T cells.
  • APCs antigen presenting cells
  • Costimulatory signals stimulate antigen-specific T cell proliferation and differentiate into effector T cells. In the absence of costimulatory signals, T cells will enter a state of no response or autoimmune tolerance, and even enter programmed death.
  • OX40 is a member of the TNFR receptor superfamily and is a type I transmembrane glycoprotein. OX40 is predominantly expressed on activated CD4 + T cells and CD8 + T cells (Paterson et al. (1987) Mol Immunol 24: 1281-1290). The extracellular domain of OX40 consists of three cysteine rich domains (CRD) and one C-terminal incomplete CRD (Deanne M et al. (2006) Structure 14: 1321-1330). OX40 is a secondary costimulatory molecule. Unlike CD28, OX40 is not expressed on the surface of resting T cells, but is highly expressed in T-cell activation for 24-72 hours.
  • OX40 ligand OX40L (TNFSF4, TXGP1, OX-40L, gp34 or CD252) is a type II transmembrane glycoprotein expressed in activated antigen-presenting cells such as dendritic cells, B cells, etc. (Godfrey, WR et al ( 1994) JExpMed 180: 757–762). OX40/OX40L signaling plays a very important role in the activation, proliferation and inhibition of apoptosis of T cells.
  • activated OX40 antibodies have been used to screen and produce activated OX40 antibodies.
  • the application provides an isolated OX40 antigen polypeptide.
  • the OX40 antigen polypeptide of the present application is capable of specifically binding to an OX40 agonistic antibody and can be used to produce the OX40 agonistic antibody.
  • the OX40 agonistic antibody is capable of preventing the formation of OX40-OX40L hexamers.
  • the OX40 antigen polypeptides of the present application can be used to detect, recognize and/or screen OX40 agonistic antibodies.
  • the OX40 antigen polypeptides of the present application can also be used to prepare vaccines, such as cancer vaccines.
  • the OX40 antigen polypeptides of the present application can be used to prevent and/or treat cancer.
  • the application provides an isolated OX40 antigen polypeptide, which can comprise the amino acid sequences set forth in any one or more of SEQ ID NOs: 1-32 and fragments or variants thereof.
  • the variant of the amino acid sequence may comprise: 1) at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92) of the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequence of sequence homology; and/or 2) in SEQ ID NO:
  • One or more of the amino acid sequences shown in any one of 1-32 are substituted, deleted or added (for example, 1-2, 1-3, 1-4, 1-5, 1) Amino acid sequences obtained by -6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 or more amino acids.
  • the variant of the amino acid sequence does not comprise the mutation G70A and/or the mutation F71A. In certain embodiments, the variant of the amino acid sequence does not comprise an amino acid mutation at residue G70 and/or residue F71.
  • the residue G70 means the residue G corresponding to the 70th position in the amino acid sequence shown by SEQ ID NO: 33
  • the residue F71 means the 71st position corresponding to the amino acid sequence shown by SEQ ID NO: Residue F.
  • the mutation G70A means that the residue G corresponding to the 70th position in the amino acid sequence shown by SEQ ID NO: 33 is mutated to A.
  • the mutation F71A means that the residue F corresponding to the 71st position in the amino acid sequence shown by SEQ ID NO: 33 is mutated to A.
  • residue Xn refers to the residue X corresponding to the n-th position in the amino acid sequence shown by SEQ ID NO: 33, wherein n is a positive integer and X is an abbreviation of any amino acid residue.
  • the mutation "XnY” means that the residue X corresponding to the n-th position in the amino acid sequence shown by SEQ ID NO: 33 is mutated to Y, wherein n is a positive integer, and X and Y are each independently an arbitrary amino acid. Abbreviation for residue, and X is different from Y.
  • a residue in an amino acid sequence "corresponds to" a residue in another amino acid sequence generally refers to a residue corresponding to the amino acid sequence alignment under optimized conditions.
  • the sequence alignment can be performed by methods known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, and the like.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • NEEDLE Megalign
  • Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment in the full length sequence being compared.
  • the residues at positions 1 to 7 in the amino acid sequence shown by SEQ ID NO: 2 correspond to residues 65 to 71 of the amino acid sequence shown in SEQ ID NO: 33, respectively.
  • the isolated OX40 antigen polypeptides described herein comprise from about 4 to 160 (eg, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, At least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75 At least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160 or more) Amino acid residues.
  • at least 5 eg, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, At least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75 At least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least
  • an isolated OX40 antigen polypeptide described herein is derived from an extracellular domain of OX40, a fragment or variant thereof.
  • it can be derived from the extracellular domain of human, cynomolgus, mouse or rat OX40, fragments or variants thereof.
  • the isolated OX40 antigen polypeptides described herein are derived from human OX40.
  • it may be derived from the extracellular domain of human OX40, a fragment or variant thereof.
  • the amino acid sequence of the human OX40 extracellular domain can be as set forth in SEQ ID NO:43.
  • an isolated OX40 antigen polypeptide of the present application is derived from a region of an OX40 (eg, human OX40) extracellular domain comprising one or more domains or fragments thereof in the lower panel: cysteine rich Set domain 1 (CRD1), cysteine rich domain 2 (CRD2), and cysteine rich domain 3 (CRD3).
  • an isolated OX40 antigen polypeptide of the present application is derived from a region of an OX40 (eg, human OX40) extracellular domain comprising one or more domains or fragments thereof in the lower panel: cysteine rich Set domain 1 (CRD1) and cysteine rich domain 2 (CRD2).
  • an isolated OX40 antigen polypeptide of the present application does not comprise the following region of the OX40 (eg, human OX40) extracellular domain: cysteine rich domain 3 (CRD3) or cysteine rich Set Domain 4 (CRD4).
  • cysteine rich domain 3 CCD3
  • cysteine rich Set Domain 4 CCD4
  • an isolated OX40 antigen polypeptide of the present application can comprise a region derived from one or more domains or fragments thereof in the lower panel: 1) module A1 of CRD1 of OX40 (eg, human OX40); 2) OX40 (eg, , module OX2 of CRD1 of human OX40); 3) module A1 of CRD2 of OX40 (eg, human OX40); and module B2 of CRD2 of OX40 (eg, human OX40).
  • the amino acid sequence of module A1 in CRD1 of human OX40 can be as shown in SEQ ID NO: 45
  • the amino acid sequence of module B2 in CRD1 of human OX40 can be as shown in SEQ ID NO: 46
  • the amino acid sequence of CRD1 of human OX40 can be As shown in SEQ ID NO:47
  • the amino acid sequence of the module A1 in the CRD2 of human OX40 can be as shown in SEQ ID NO: 48
  • the amino acid sequence of the module B2 in the CRD2 of human OX40 can be as shown in SEQ ID NO: 49
  • the amino acid sequence of CRD2 of human OX40 can be as SEQ. ID NO: 50 is shown.
  • amino acid sequence of CRD3 of human OX40 can be as shown in SEQ ID NO:51.
  • amino acid sequence of CRD4 of human OX40 can be as shown in SEQ ID NO:52.
  • an isolated OX40 antigen polypeptide of the present application is immunogenic.
  • the application provides a fusion protein or immunoconjugate comprising an isolated OX40 antigen polypeptide as described herein.
  • the application provides one or more isolated nucleic acid molecules encoding an isolated OX40 antigen polypeptide described herein and/or a fusion protein or immunoconjugate as described herein.
  • the application provides one or more vectors that can comprise one or more of the isolated nucleic acid molecules described herein.
  • the application provides one or more cells (eg, host cells) or non-human organisms, which can comprise or express an OX40 antigen polypeptide described herein, a fusion protein or immunoassay as described herein a compound, one or more isolated nucleic acid molecules described herein, and/or one or more vectors described herein.
  • cells eg, host cells
  • non-human organisms which can comprise or express an OX40 antigen polypeptide described herein, a fusion protein or immunoassay as described herein a compound, one or more isolated nucleic acid molecules described herein, and/or one or more vectors described herein.
  • the application provides a composition (eg, a pharmaceutical composition), which can comprise an OX40 antigen polypeptide as described herein, a fusion protein or immunoconjugate as described herein, as described herein A nucleic acid molecule, a vector described herein, or a cell (e.g., a host cell) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • a composition eg, a pharmaceutical composition
  • a composition eg, a pharmaceutical composition
  • a composition which can comprise an OX40 antigen polypeptide as described herein, a fusion protein or immunoconjugate as described herein, as described herein
  • a nucleic acid molecule e.g., a vector described herein, or a cell (e.g., a host cell) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the application provides a vaccine, which can comprise one or more substances selected from the group consisting of: and optionally an immunogenic carrier: 1) one or more OX40 antigen polypeptides described herein 2) a fusion protein or immunoconjugate as described herein; 3) a nucleic acid molecule as described herein; 4) a vector as described herein; 5) a cell (eg, a host cell) as described herein; And 6) a composition (e.g., a pharmaceutical composition) as described herein.
  • the vaccine is a cancer vaccine.
  • the application provides an antibody that is capable of specifically binding to an OX40 antigen polypeptide as described herein.
  • the antibody can be an OX40 agonistic antibody.
  • the antibody is a monoclonal antibody.
  • the antibody does not comprise DF004.
  • the antibody does not substantially bind to an OX40 (eg, human OA40) variant comprising the mutant G70A and/or the mutant F71A.
  • the antibody does not substantially bind to a human OX40 variant having the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO:36.
  • the application provides a pharmaceutical composition that can comprise an antibody (eg, an OX40 agonistic antibody) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • an antibody eg, an OX40 agonistic antibody
  • the application provides a method for producing an OX40 agonistic antibody.
  • the method can comprise immunizing an animal with an OX40 antigen polypeptide as described herein.
  • the method can further comprise isolating an antibody that specifically binds to the OX40 antigen polypeptide.
  • the application provides methods for screening and/or detecting OX40 agonistic antibodies.
  • the method can comprise allowing a candidate antibody or antigen-binding fragment thereof to be screened and/or to be detected, or a cell or cell secretion expressing the candidate antibody or antigen-binding fragment thereof, and an OX40 antigen polypeptide or present described herein.
  • the fusion protein or immunoconjugate described in the application is contacted.
  • the method can also include determining whether the candidate antibody or antigen-binding fragment thereof specifically binds to the OX40 antigen polypeptide, and when the specific binding is detected, identifying the candidate antibody as an OX40 agonistic antibody.
  • the OX40 agonistic antibody can include DF004.
  • the application provides the use of an OX40 antigenic polypeptide described herein or a fusion protein or immunoconjugate as described herein for the preparation of an immunologically active substance, which may comprise an OX40 agonistic antibody .
  • the application provides the use of an OX40 antigen polypeptide described herein, a fusion protein or immunoconjugate described herein, or an antibody described herein for the preparation of a medicament, the medicament being useful for prevention And / or treat cancer.
  • the application provides a method of preventing the formation of OX40-OX40L hexamer, the method comprising administering an agent that specifically binds to an OX40 antigen polypeptide described herein.
  • an agent that specifically binds to an OX40 antigen polypeptide described herein can be an agonist of OX40.
  • the agent that specifically binds to an OX40 antigen polypeptide described herein is an OX40 agonistic antibody.
  • the application provides the use of the OX40 antigen polypeptide or a fusion protein or immunoconjugate described herein for the preparation of a reagent useful for preventing the formation of OX40-OX40L hexamer.
  • the agent is capable of activating OX40 as an agonist.
  • the agent can be an OX40 agonistic antibody.
  • the application provides the use of the OX40 antigen polypeptide or a fusion protein or immunoconjugate described herein for the preparation of a reagent useful for detecting, identifying and/or screening OX40 agonistic antibodies .
  • the OX40 agonistic antibody can comprise DF004.
  • the application provides methods of preventing and/or treating cancer in a subject.
  • the method can comprise administering to the subject an effective amount of one or more substances selected from the group consisting of: 1) an OX40 antigen polypeptide as described herein; 2) a fusion protein or immunization described herein a conjugate; 3) a nucleic acid molecule as described herein; 4) a vector as described herein; 5) a cell (eg, a host cell) as described herein; 6) a pharmaceutical composition as described herein; The vaccine described herein.
  • Figure 1 shows the results of amino acid sequence alignment of OX40 fragments of different species.
  • Figure 2 shows the complex formed by OX40 and its ligand OX40L, and the complex formed by OX40 and its agonistic antibody.
  • Figure 3 shows the OX40 epitope sequence that specifically binds to an OX40 agonistic antibody, in which the major amino acid residues involved in the binding are indicated.
  • Figure 4 shows the effect of mutations in the amino acid sequence in the OX40 epitope polypeptide on its binding to antibodies.
  • Figure 5 shows the results of kinetic parameter detection of binding of OX40 epitope polypeptides at different positions to antibodies.
  • Figures 6A-6G show the results of kinetic curve fitting of binding of OX40 epitope polypeptides at different positions to antibodies.
  • an antibody generally refers to a polypeptide molecule that is capable of specifically recognizing and/or neutralizing a particular antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule or fragment comprising its antigen-binding portion.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives including, but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (eg, scFv), and antibody fragments that bind to an antigen (eg, , Fab, Fab' and (Fab) 2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further distinguished as hypervariable regions termed complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CRD1, FR2, CRD2, FR3, CRD3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the term "antigen-binding portion” generally refers to one or more fragments of an antibody that function to specifically bind antigen.
  • the antigen binding function of an antibody can be achieved by a full length fragment of the antibody.
  • the antigen binding function of an antibody can also be achieved by the following fragments: (1) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH domains; (2) a F(ab')2 fragment, including a bivalent fragment of a disulfide-linked two Fab fragment at the hinge region; (3) an Fd fragment consisting of a VH and a CH domain; (4) an Fv fragment consisting of a VL and VH domain of an antibody single arm; (5) a dAb fragment consisting of a VH domain (Ward et al.
  • the "antigen-binding portion” may further include a monovalent single-chain molecule Fv (scFv) formed by pairing of VL and VH (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883).
  • scFv monovalent single-chain molecule Fv
  • the "antigen-binding portion” may further comprise an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) An immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • OX40 generally refers to an OX40 protein, which may include any OX40 protein expressed by a cell which is naturally or genetically engineered to contain an OX40 protein gene, and variants, functional fragments thereof, isoforms thereof (isoform) And species homologs.
  • OX40 is a type I transmembrane glycoprotein that functions by forming a multimer (eg, a hexamer) by interacting with its ligand OX40L.
  • the term "OX40” can include a polypeptide having at least 80% amino acid sequence identity to UniProt accession number P43489.1 and specifically binding to OX40L or a fragment thereof.
  • OX40 as used in the present application includes human OX40 (hOX40), variants, isoforms and interspecies homologs of hOX40, and analogs thereof having at least one co-epitope with hOX40.
  • OX40 protein of the present application can be a human OX40 protein.
  • the human OX40 protein may comprise an amino acid sequence as set forth in SEQ ID NO: xxx or a variant of the amino acid sequence, for example, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% variant of sequence homology.
  • the term "monoclonal antibody” generally refers to an antibody that is highly uniform, produced only by a single B cell clone, and that is directed against a particular antigenic epitope.
  • Monoclonal antibodies display specificity and affinity for an epitope.
  • a monoclonal antibody can be produced by a hybridoma which is a hybridoma cell obtained by fusing a B cell having the ability to secrete a specific antibody and a myeloma cell having an infinite ability to reproduce.
  • epitope generally refers to an antigenic determinant, ie, a portion of a molecule that is recognized by the immune system (eg, recognized by an antibody).
  • an epitope can be a discrete three-dimensional site on an antigen that is recognized by the immune system.
  • An epitope typically consists of a chemically active surface group of a molecule (eg, an amino acid or a sugar side chain) and typically has specific three dimensional structural characteristics as well as specific charge characteristics. Epitopes can be classified into conformational epitopes and non-conformational epitopes (linear epitopes) depending on the structure.
  • a conformational epitope differs from a non-conformational epitope in that the former loses binding in the presence of a denaturing solvent, while the latter does not.
  • An epitope that is only on the surface of an antigenic substance and that readily binds to an antigen recognition receptor or antibody may be referred to as a functional epitope; an epitope that is located inside the molecule and is not immunogenic may be referred to as a hidden epitope.
  • extracellular domain generally refers to the extracellular domain of a protein.
  • the extracellular domain of the OX40 protein can generally be in the form of an OX40 polypeptide that is substantially free of transmembrane and cytoplasmic domains.
  • the definition of transmembrane is identified according to conventional standards used in the art to identify the type of hydrophobic domain.
  • the exact boundaries of the transmembrane domains can vary, but most are similar.
  • the extracellular domain of the OX40 protein can contain about 5 (or 4, 3, 2 or 1) amino acids on either side of the transmembrane domain or extracellular domain boundaries.
  • the extracellular domain of the OX40 protein can carry an associated signal peptide.
  • isolated nucleic acid molecule or isolated nucleic acid generally refers to an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length isolated or artificially synthesized from its natural environment. Glyceric acid or an analogue thereof.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a plurality of the above functions.
  • the vector may be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
  • the term "host cell” generally refers to a plasmid or vector that may or already contains a nucleic acid molecule as described herein, or an individual cell, cell line or cell culture capable of expressing an antigenic polypeptide as described herein.
  • the host cell can comprise a progeny of a single host cell. Due to natural, unexpected or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genomic to the original parental cell, but are capable of expressing the antigenic polypeptides described herein.
  • the host cell can be obtained by transfecting cells in vitro using a vector described herein.
  • the host cell may be a prokaryotic cell (such as E.
  • the host cell is a mammalian cell.
  • the term "recombinant host cell” generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell but also the progeny of these cells.
  • tumor generally refers to a neoplasm formed by the proliferation of local tissue cells by a body (eg, a cell or a component thereof) in a mammal under the action of various tumorigenic factors.
  • the tumor may include lymphoma, blastoma, sarcoma, hematoma, and the like.
  • pharmaceutically acceptable adjuvant generally means any solvent, dispersion medium, coating, isotonic and absorption delaying agent, etc., excipients compatible with the active ingredient to be applied. Or other drug carrier.
  • the term “subject” generally refers to any human or non-human animal.
  • non-human animal may include all vertebrates, such as mammals and non-mammals, such as non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.
  • the term "about” generally means a range of 0.5% to 10% above or below a specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5% above or below a specified value, Variations within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • OX40 antigen polypeptide In the present application, the terms “OX40 antigen polypeptide”, “OX40 epitope polypeptide”, “OX40 polypeptide”, “OX40 epitope” and the like are used interchangeably and generally refer to a polypeptide derived from an OX40 protein (eg, human OX40). A fragment that is capable of being recognized by an OX40-specific antibody, binds to an OX40-specific antibody, and/or is capable of producing an OX40-specific antibody.
  • OX40 antigen polypeptide may consist of a contiguous amino acid residue on the OX40 protein, and may also refer to a combination of amino acid residues located at discrete positions on the OX40 protein.
  • the term “immunogenicity” generally refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes.
  • the antigenic polypeptide is capable of stimulating specific immune cells, activating, proliferating, and differentiating immune cells, ultimately producing characteristics of immune effector substances (eg, antibodies) and/or sensitized lymphocytes.
  • the term “immunogenicity” may also refer to a specific immune response in which the body's immune system is capable of forming antibodies or sensitizing T lymphocytes after the antigenic polypeptide has stimulated the body.
  • non-human objects generally refers to life forms other than humans.
  • non-human objects may include plants, animals, microorganisms, and/or viruses, etc., or portions of their vital features.
  • the microorganism can be, for example, a bacterium.
  • the animal can be, for example, a vertebrate such as a mammal.
  • non-human organisms include non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, rats, mice, and the like.
  • the term "vaccine” generally refers to a disease or condition made with various types of immunogens (eg, pathogens or antigenic polypeptide fragments, etc.) for vaccination and/or treatment that is directly or indirectly related to the immunogen.
  • immunogens eg, pathogens or antigenic polypeptide fragments, etc.
  • cancer vaccine generally refers to a biological product that activates a subject's immune system against cancer by utilizing tumor cell-associated antigens (eg, antigenic polypeptides or combinations thereof).
  • tumor cell-associated antigens eg, antigenic polypeptides or combinations thereof.
  • an "agonistic antibody” generally refers to an antibody molecule or antigen-binding fragment thereof which is capable of activating a target molecule and/or enhancing its activity.
  • an "agonistic antibody” in the present application may be an antibody molecule or an antigen-binding fragment thereof that specifically binds to a target molecule, wherein binding of the antibody molecule or antigen-binding fragment thereof to a target molecule causes the target molecule to be in an inactive state Transition to an activated state, or from a low active state to a highly active state.
  • OX40 agonistic antibody generally refers to an antibody or antigen-binding fragment thereof capable of specifically recognizing and/or binding to OX40, and the antibody or antigen-binding fragment thereof specifically recognizes and/or binds OX40 to enable OX40 transitions from an unactivated state to an activated state, or from a low active state to a highly active state.
  • the term "immunologically active substance” generally refers to a substance capable of generating an immune response or capable of causing a specific immune response in the body.
  • the immunologically active substance can be produced by effector cells (e.g., T cells) or other cells.
  • Common immunologically active substances may include, for example, antibodies or antigen-binding fragments thereof, chemokines, growth factors, immunological adjuvants, cytokines (eg, interferons, interleukins, lymphokines, etc.), cytokines, inflammatory factors, viruses, or Its components, bacteria or components thereof.
  • the immune response or specific immune response can include, for example, enhancing antibody production, enhancing humoral responses, enhancing cellular immune responses, and the like.
  • OX40-OX40L hexamer generally refers to a protein hexamer composed of OX40 and its ligand OX40L.
  • three OX40 protein molecules and three OX40L protein molecules may be included in the OX40-OX40L hexamer.
  • each OX40 protein molecule binds to two OX40L protein molecules, and each OX40L protein molecule binds to two OX40 protein molecules.
  • the OX40 protein molecule is alternately aligned with the OX40L protein molecule to form the OX40-OX40L hexamer.
  • the term "agonist” generally refers to a substance or agent that is capable of activating a target molecule and/or enhancing its activity.
  • an "agonist” in the present application may be an agonistic antibody or other substance which can convert a target molecule from an unactivated state to an activated state by direct or indirect binding to a target molecule, or by low activity. The state changes to a highly active state.
  • "OX40 agonist” generally refers to a substance or agent that is capable of activating OX40 and/or enhancing its activity.
  • an OX40 agonist can convert OX40 from an unactivated state to an activated state, or from a low active state to a highly active state, by binding directly or indirectly to OX40.
  • the term "preventing OX40-OX40L hexamer formation” generally means that a hexamer structure cannot be formed between OX40 and its ligand OX40L. In some cases, OX40 and OX40L are still capable of binding to each other, but are unable to form hexamers (eg, only dimers, trimers, tetramers, etc.). In some cases, OX40 and OX40L cannot bind to each other or bind to an active molecular conformation.
  • sequence homology generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences.
  • the default parameter settings can be used when using computer programs or software (eg, Emboss Needle or BestFit) to determine sequence identity, similarity, or homology between different amino acid sequences. You can also choose an appropriate scoring matrix, such as blosum45 or blosum80, to optimize identity, similarity, or homology scores.
  • a homologous polynucleotide comprises a polynucleotide that is capable of hybridizing to a control polynucleotide sequence under stringent conditions and having at least 60%, at least 65% compared to a control polynucleotide sequence. At least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even at least 100% sequence identity.
  • a homologous polypeptide can be a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% of the control polypeptide sequence when subjected to sequence alignment under optimized conditions. At least 99%, or even at least 100% sequence identity.
  • sequence alignments can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, and the like. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment in the full length sequence being compared.
  • fusion protein generally refers to a polypeptide molecule in which the amino acid sequence of one polypeptide (eg, an OX40 antigen polypeptide of the present application) is directly or indirectly (eg, via a linker) and another heterologous source.
  • the amino acid sequences of the polypeptides are fused (eg, by peptide bonds).
  • immunoconjugate generally refers to a method by conjugating a polypeptide (eg, an OX40 antigenic polypeptide of the present application) to an antibody or fragment thereof (eg, an antibody constant region or an antibody Fc region). Protein molecules.
  • a polypeptide eg, an OX40 antigenic polypeptide of the present application
  • an antibody or fragment thereof eg, an antibody constant region or an antibody Fc region.
  • OX40 antigen polypeptide OX40 antigen polypeptide, fusion protein or immunoconjugate
  • the application provides an isolated OX40 antigen polypeptide.
  • An isolated OX40 antigen polypeptide as described herein may comprise the amino acid sequences set forth in any one or more of SEQ ID NOs: 1-32 and fragments or variants thereof.
  • the variant of the amino acid sequence may comprise: 1) at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92) of the amino acid sequence set forth in any one of SEQ ID NOs: 1-32.
  • amino acid sequence of sequence homology and/or 2) in SEQ ID NO:
  • amino acid sequences shown in any one of 1-32 are substituted, deleted or added (for example, 1-2, 1-3, 1-4, 1-5, 1) Amino acid sequences obtained by -6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 or more amino acids.
  • the substitution of the amino acid may be a conservative amino acid substitution or a non-conservative amino acid substitution.
  • the substituted OX40 antigen polypeptide is still capable of being recognized or bound (eg, specifically recognized or bound) by an OX40 agonistic antibody (eg, DF004 or an antigen binding fragment thereof), or is still capable of producing an OX40 agonistic antibody (eg, An antibody that has one or more characteristics of DF004 or an antigen binding fragment thereof.
  • the amino acid substitutions can be non-conservative substitutions.
  • the non-conservative substitutions can include altering amino acid residues in a protein or polypeptide of interest in a non-conservative form, such as changing amino acid residues having a certain side chain size or a certain property (eg, hydrophilicity) to be different. Amino acid residues of side chain size or different properties (eg, hydrophobicity).
  • amino acid substitutions can also be conservative substitutions.
  • the conservative substitutions may include altering amino acid residues in a protein or polypeptide of interest in a conservative form, such as changing amino acid residues having a certain side chain size or a certain property (eg, hydrophilicity) to have the same or similar Amino acid residues of side chain size or of the same or similar properties (eg, still hydrophilic).
  • Such conservative substitutions generally do not have a large impact on the structure or function of the protein produced.
  • amino acid sequence variants of the isolated OX40 antigen polypeptide can include conservative amino acid substitutions that do not significantly alter the structure of the protein or its function (eg, the ability to produce or bind to an OX40 agonistic antibody).
  • Amino acid group with non-polar side chains alanine, valine, leucine, isoleucine, valine, phenylalanine, tryptophan and methionine.
  • Uncharged, amino acid group with polar side chains glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • Positively charged basic amino acids lysine, arginine and histidine.
  • Amino acids with phenyl groups phenylalanine, tryptophan and tyrosine.
  • the variant of the amino acid sequence does not comprise the mutation G70A and/or the mutation F71A. In certain embodiments, the variant of the amino acid sequence does not comprise an amino acid mutation at residue G70 and/or residue F71.
  • the residue G70 means the residue G corresponding to the 70th position in the amino acid sequence shown by SEQ ID NO: 33
  • the residue F71 means the 71st position corresponding to the amino acid sequence shown by SEQ ID NO: Residue F.
  • the mutation G70A means that the residue G corresponding to the 70th position in the amino acid sequence shown by SEQ ID NO: 33 is mutated to A.
  • the mutation F71A means that the residue F corresponding to the 71st position in the amino acid sequence shown by SEQ ID NO: 33 is mutated to A.
  • the OX40 antigen polypeptide may be derived from OX40 (eg, human OX40), and it does not comprise an amino acid mutation at residue G corresponding to position 70 in the amino acid sequence set forth in SEQ ID NO:33.
  • the OX40 antigen polypeptide may be derived from OX40 (eg, human OX40), and it does not comprise an amino acid mutation at residue F corresponding to position 71 of the amino acid sequence set forth in SEQ ID NO:33.
  • a residue in an amino acid sequence "corresponds to" a residue in another amino acid sequence generally refers to a residue corresponding to the amino acid sequence alignment under optimized conditions.
  • the sequence alignment can be performed by methods known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, and the like.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • NEEDLE Megalign
  • Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment in the full length sequence being compared.
  • the residues at positions 1 to 7 in the amino acid sequence shown by SEQ ID NO: 2 correspond to residues 65 to 71 of the amino acid sequence shown in SEQ ID NO: 33, respectively.
  • an isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: X 1 PCX 2 X 3 GF (SEQ ID NO: 1), wherein X 1 can be R, A or H, and X 2 can be G Or E, and X 3 may be P or T.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence RPCGPGF (SEQ ID NO: 2).
  • an isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: X 1 PCX 2 X 3 GFYNX 4 X 5 VX 6 X 7 X 8 X 9 C (SEQ ID NO: 3), wherein X 1 can Is R, A or H, X 2 may be G or E, X 3 may be P or T, X 4 may be D or E, X 5 may be V or A, X 6 may be S or N, and X 7 may Is S or Y, X 8 may be K or D, and X 9 may be P or T.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence RPCGPGFYNDVVSSKPC (SEQ ID NO: 4).
  • an isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: X 1 PCX 2 X 3 GFYNX 4 X 5 VX 6 X 7 X 8 X 9 CKX 10 CTX 11 CN (SEQ ID NO: 5), Wherein X 1 may be R, A or H, X 2 may be G or E, X 3 may be P or T, X 4 may be D or E, X 5 may be V or A, and X 6 may be S or N X 7 may be S or Y, X 8 may be K or D, X 9 may be P or T, X 10 may be P, A or Q, and X 11 may be W, A or Q.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence RPCGPGFYNDVVSSKPCKPCTWCN (SEQ ID NO: 6).
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: X 1 PCX 2 X 3 GFYNX 4 X 5 VX 6 X 7 X 8 X 9 CKX 10 CTX 11 CNX 12 RSGSEX 13 KQX 14 CTX 15 TX 16 DTVC (SEQ ID NO: 7), wherein X 1 may be R, A or H, X 2 may be G or E, X 3 may be P or T, X 4 may be D or E, and X 5 may be V Or A, X 6 may be S or N, X 7 may be S or Y, X 8 may be K or D, X 9 may be P or T, X 10 may be P, A or Q, and X 11 may be W , A or Q, X 12 may be L or H, X 13 may be R or L, X 14 may be L or N, X 15 may be A or P, and X 16 may be Q or E.
  • an isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: GMVSRCX 1 X 2 X 3 X 4 X 5 TX 6 CX 7 PCX 8 X 9 GF (SEQ ID NO: 9), wherein X 1 can Is S or D, X 2 may be R or H, X 3 may be S or T, X 4 may be Q or R, X 5 may be N or D, X 6 may be V or L, and X 7 may be R , A or H, X 8 may be G or E, and X 9 may be P or T.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence GMVSRCSRSQNTVCRPCGPGF (SEQ ID NO: 10).
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: CX 1 ECX 2 PGX 3 GMVSRCX 4 X 5 X 6 X 7 X 8 TX 9 CX 10 PCX 11 X 12 GF (SEQ ID NO: 11 Wherein X 1 may be H or R, X 2 may be R or Q, X 3 may be N or H, X 4 may be S or D, X 5 may be R or H, and X 6 may be S or T X 7 may be Q or R, X 8 may be N or D, X 9 may be V or L, X 10 may be R, A or H, X 11 may be G or E, and X 12 may be P or T.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence CHECRPGNGMVSRCSRSQNTVCRPCGPGF (SEQ ID NO: 12).
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: LX 1 CVX 2 X 3 TYPSX 4 X 5 X 6 CCX 7 ECX 8 PGX 9 GMVSRCX 10 X 11 X 12 X 13 X 14 TX 15 CX 16 PCX 17 X 18 GF (SEQ ID NO: 13), wherein X 1 may be H or N, X 2 may be G or K, X 3 may be D or H, X 4 may be N or G, and X 5 may Is D or H, X 6 may be R or K, X 7 may be H or R, X 8 may be R or Q, X 9 may be N or H, X 10 may be S or D, and X 11 may be R Or H, X 12 may be S or T, X 13 may be Q or R, X 14 may be N or D, X 15 may be V or L, X 16 may be R, A or H, and X 17 may be G
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: GMVSRCX 1 X 2 X 3 X 4 X 5 TX 6 CX 7 PCX 8 X 9 GFYNX 10 X 11 VX 12 X 13 X 14 X 15 C (SEQ ID NO: 15), wherein X 1 may be S or D, X 2 may be R or H, X 3 may be S or T, X 4 may be Q or R, and X 5 may be N or D, X 6 may be V or L, X 7 may be R, A or H, X 8 may be G or E, X 9 may be P or T, X 10 may be D or E, and X 11 may be V or A, X 12 may be S or N, X 13 may be S or Y, X 14 may be K or D, and X 15 may be P or T.
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence GMVSRCSRSQNTV
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: CX 1 ECX 2 PGX 3 GMVSRCX 4 X 5 X 6 X 7 X 8 TX 9 CX 10 PCX 11 X 12 GFYNX 13 X 14 VX 15 X 16 X 17 X 18 C (SEQ ID NO: 17), wherein X 1 may be H or R, X 2 may be R or Q, X 3 may be N or H, X 4 may be S or D, and X 5 may Is R or H, X 6 may be S or T, X 7 may be Q or R, X 8 may be N or D, X 9 may be V or L, X 10 may be R, A or H, X 11 may Is G or E, X 12 may be P or T, X 13 may be D or E, X 14 may be V or A, X 15 may be S or N, X 16 may be S or Y, and X 17 may be K
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: LX 1 CVX 2 X 3 TYPSX 4 X 5 X 6 CCX 7 ECX 8 PGX 9 GMVSRCX 10 X 11 X 12 X 13 X 14 TX 15 CX 16 PCX 17 X 18 GFYNX 19 X 20 VX 21 X 2 2 X 23 X 24 C (SEQ ID NO: 19), wherein X 1 may be H or N, X 2 may be G or K, and X 3 may be D or H, X 4 may be N or G, X 5 may be D or H, X 6 may be R or K, X 7 may be H or R, X 8 may be R or Q, and X 9 may be N or H, X 10 may be S or D, X 11 may be R or H, X 12 may be S or T, X 13 may be Q or R, X 14 may be N or D, and X 15 may be V
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: GMVSRCX 1 X 2 X 3 X 4 X 5 TX 6 CX 7 PCX 8 X 9 GFYNX 10 X 11 VX 12 X 13 X 14 X 15 CKX 16 CTX 17 CN (SEQ ID NO: 21), wherein X 1 may be S or D, X 2 may be R or H, X 3 may be S or T, X 4 may be Q or R, and X 5 may be N Or D, X 6 may be V or L, X 7 may be R, A or H, X 8 may be G or E, X 9 may be P or T, X 10 may be D or E, and X 11 may be V Or A, X 12 may be S or N, X 13 may be S or Y, X 14 may be K or D, X 15 may be P or T, X 16 may be P, A or Q, and X 17 may be W, A
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: CX 1 ECX 2 PGX 3 GMVSRCX 4 X 5 X 6 X 7 X 8 TX 9 CX 10 PCX 11 X 12 GFYNX 13 X 14 VX 15 X 16 X 17 X 18 CKX 19 CTX 20 CN (SEQ ID NO: 23), wherein X 1 may be H or R, X 2 may be R or Q, X 3 may be N or H, and X 4 may be S or D X 5 may be R or H, X 6 may be S or T, X 7 may be Q or R, X 8 may be N or D, X 9 may be V or L, and X 10 may be R, A or H X 11 may be G or E, X 12 may be P or T, X 13 may be D or E, X 14 may be V or A, X 15 may be S or N, and X 16 may be S or Y,
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: LX 1 CVX 2 X 3 TYPSX 4 X 5 X 6 CCX 7 ECX 8 PGX 9 GMVSRCX 10 X 11 X 12 X 13 X 14 TX 15 CX 16 PCX 17 X 18 GFYNX 19 X 20 VX 21 X 2 2 X 23 X 24 CKX 25 CTX 26 CN (SEQ ID NO: 25), wherein X 1 may be H or N, and X 2 may be G or K, X 3 It may be D or H, X 4 may be N or G, X 5 may be D or H, X 6 may be R or K, X 7 may be H or R, X 8 may be R or Q, and X 9 may be N or H, X 10 may be S or D, X 11 may be R or H, X 12 may be S or T, X 13 may be Q or R, X 14 may be N or
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: GMVSRCX 1 X 2 X 3 X 4 X 5 TX 6 CX 7 PCX 8 X 9 GFYNX 10 X 11 VX 12 X 13 X 14 X 15 CKX 16 CTX 17 CNX 18 RSGSEX 19 KQX 20 CTX 2 1 TX 22 DTVC (SEQ ID NO: 27), where X 1 may be S or D, X 2 may be R or H, and X 3 may be S or T, X 4 It may be Q or R, X 5 may be N or D, X 6 may be V or L, X 7 may be R, A or H, X 8 may be G or E, and X 9 may be P or T, X 10 It may be D or E, X 11 may be V or A, X 12 may be S or N, X 13 may be S or Y, X 14 may be K or D, X 15 may be P or
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: CX 1 ECX 2 PGX 3 GMVSRCX 4 X 5 X 6 X 7 X 8 TX 9 CX 10 PCX 11 X 12 GFYNX 13 X 14 VX 15 X 16 X 17 X 18 CKX 19 CTX 20 CNX 21 RSG SEX 22 KQX 23 CTX 24 TX 25 DTVC (SEQ ID NO: 29), where X 1 may be H or R, X 2 may be R or Q, and X 3 may be N or H, X 4 may be S or D, X 5 may be R or H, X 6 may be S or T, X 7 may be Q or R, X 8 may be N or D, and X 9 may be V or L, X 10 may be R, A or H, X 11 may be G or E, X 12 may be P or T, X 13 may be D or E, X 14 may be V or A,
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence CHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC (SEQ ID NO: 30).
  • the isolated OX40 antigen polypeptide of the present application comprises an amino acid sequence: LX 1 CVX 2 X 3 TYPSX 4 X 5 X 6 CCX 7 ECX 8 PGX 9 GMVSRCX 10 X 11 X 12 X 13 X 14 TX 15 CX 16 PCX 17 X 18 GFYNX 19 X 20 VX 21 X 2 2 X 23 X 24 CKX 25 CTX 26 CNX 27 RSGSEX 28 KQX 29 CTX 30 TX 31 DTVC (SEQ ID NO: 31), where X 1 can be H or N, X 2 may be G or K, X 3 may be D or H, X 4 may be N or G, X 5 may be D or H, X 6 may be R or K, and X 7 may be H or R, X 8 It may be R or Q, X 9 may be N or H, X 10 may be S or D, X 11 may be R or H, X 12 may be S or
  • the isolated OX40 antigen polypeptide can comprise the amino acid sequence LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC (SEQ ID NO: 32).
  • the isolated OX40 antigen polypeptides described herein comprise from about 4 to 160 (eg, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, At least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75 At least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160 or more) Amino acid residues.
  • at least 5 eg, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, At least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75 At least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least
  • an isolated OX40 antigen polypeptide described herein can be derived from an extracellular domain of OX40, a fragment or variant thereof.
  • it can be derived from the extracellular domain of human, cynomolgus, mouse or rat OX40, fragments or variants thereof.
  • the isolated OX40 antigen polypeptides described herein are derived from human OX40.
  • it can be derived from the extracellular domain of human OX40, a fragment or variant thereof.
  • the amino acid sequence of the human OX40 extracellular domain can be as set forth in SEQ ID NO:43.
  • the OX40 antigen polypeptide may comprise an extracellular domain of OX40 (such as human OX40), a variant or portion thereof, such as one or more (eg, 1, 2, 3 or 4) cysteine Acid rich domain (CRD), variants or parts thereof.
  • OX40 such as human OX40
  • a variant or portion thereof such as one or more (eg, 1, 2, 3 or 4) cysteine Acid rich domain (CRD), variants or parts thereof.
  • CCD cysteine Acid rich domain
  • an isolated OX40 antigen polypeptide of the present application is derived from a region of an OX40 (eg, human OX40) extracellular domain comprising one or more domains or fragments thereof in the lower panel: cysteine rich Set domain 1 (CRD1), cysteine rich domain 2 (CRD2), and cysteine rich domain 3 (CRD3).
  • an isolated OX40 antigen polypeptide of the present application is derived from a region of an OX40 (eg, human OX40) extracellular domain comprising one or more domains or fragments thereof in the lower panel: cysteine rich Set domain 1 (CRD1) and cysteine rich domain 2 (CRD2).
  • an isolated OX40 antigen polypeptide of the present application does not comprise the following region of the OX40 (eg, human OX40) extracellular domain: cysteine rich domain 3 (CRD3) or cysteine rich Set Domain 4 (CRD4).
  • cysteine rich domain 3 CCD3
  • cysteine rich Set Domain 4 CCD4
  • the OX40 antigen polypeptide comprises a CRD2 region of an OX40 (eg, human OX40) extracellular domain, a variant thereof, or a fragment thereof. In certain embodiments, the OX40 antigen polypeptide further comprises a CRD1 region of an OX40 (eg, human OX40) extracellular domain, a variant thereof, or a fragment thereof.
  • the OX40 antigen polypeptide does not comprise a CRD3 region or a fragment thereof in the extracellular domain of OX40 (eg, human OX40). In certain embodiments, the OX40 antigen polypeptide does not comprise a CRD4 region or a fragment thereof in the extracellular domain of OX40 (eg, human OX40).
  • an isolated OX40 antigen polypeptide of the present application can comprise a region derived from one or more domains or fragments thereof in the lower panel: 1) module A1 of CRD1 of OX40 (eg, human OX40); 2) OX40 (eg, , module OX2 of CRD1 of human OX40); 3) module A1 of CRD2 of OX40 (eg, human OX40); and module B2 of CRD2 of OX40 (eg, human OX40).
  • an isolated OX40 antigen polypeptide of the present application comprises a module A1, a variant or fragment thereof of CRD1 of OX40 (eg, human OX40).
  • an isolated OX40 antigen polypeptide of the present application comprises a module B2, a variant or fragment thereof of CRD1 of OX40 (eg, human OX40).
  • an isolated OX40 antigen polypeptide of the present application comprises a module A1, a variant or fragment thereof of CRD2 of OX40 (eg, human OX40).
  • an isolated OX40 antigen polypeptide of the present application comprises a module B2, a variant or fragment thereof of CRD2 of OX40 (eg, human OX40).
  • the amino acid sequence of module A1 in CRD1 of human OX40 can be as shown in SEQ ID NO: 45
  • the amino acid sequence of module B2 in CRD1 of human OX40 can be as shown in SEQ ID NO: 46
  • the amino acid sequence of CRD1 of human OX40 can be As shown in SEQ ID NO:47
  • the amino acid sequence of the module A1 in the CRD2 of human OX40 can be as shown in SEQ ID NO: 48
  • the amino acid sequence of the module B2 in the CRD2 of human OX40 can be as shown in SEQ ID NO: 49
  • the amino acid sequence of CRD2 of human OX40 can be as SEQ. ID NO: 50 is shown.
  • amino acid sequence of CRD3 of human OX40 can be as shown in SEQ ID NO:51.
  • amino acid sequence of CRD4 of human OX40 can be as shown in SEQ ID NO:52.
  • the isolated OX40 antigen polypeptide of the present application may be immunogenic.
  • the OX40 antigen polypeptide is capable of stimulating the body to form OX40-specific antibodies and/or sensitized lymphocytes.
  • the application provides a fusion protein or immunoconjugate comprising the isolated OX40 antigen polypeptide.
  • the isolated OX40 antigen polypeptide can be fused to one or more heterologous polypeptides (eg, an antibody or a component of an antibody, or other antigenic polypeptide) to provide the fusion protein or immunoconjugate.
  • the heterologous polypeptide for fusion with the isolated OX40 antigen polypeptide may comprise a constant region or an Fc region of an antibody (eg, an IgG antibody).
  • a heterologous polypeptide for fusion with the isolated OX40 antigen polypeptide can comprise other antigenic polypeptides, such as other antigenic polypeptides derived from OX40 (eg, human OX40).
  • An isolated OX40 antigen polypeptide as described herein can be linked (eg, in-frame) or indirectly (eg, via a linker, such as a peptide linker) to the one or more heterologous polypeptides, conjugated, or Fusion.
  • the fusion protein or immunoconjugate can be prepared by methods known to those skilled in the art, for example, the fusion protein or immunoconjugate can be prepared by molecular cloning and recombinant expression of the protein.
  • the application provides one or more isolated nucleic acid molecules encoding an isolated OX40 antigen polypeptide described herein and/or a fusion protein or immunoconjugate as described herein .
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification. , for example, by fractionation by gel digestion and gel electrophoresis, or (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA techniques.
  • nucleic acids encoding the OX40 antigen polypeptide and/or the fusion protein or immunoconjugate can be prepared by a variety of methods known in the art including, but not limited to, restriction fragment manipulation Or overlap extension PCR using synthetic oligonucleotides, see Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular. Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • the application provides one or more vectors that can comprise one or more of the isolated nucleic acid molecules described herein.
  • other genes may be included in the vector, such as a marker gene that allows selection of the vector in a suitable host cell and under appropriate conditions.
  • the vector may further comprise an expression control element that allows the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically comprises 5' non-transcribed sequences and 5' and 3' untranslated sequences, respectively, involved in transcriptional and translational initiation, such as TATA boxes, plus Cap sequence, CAAT sequence, and the like.
  • a 5' non-transcribed expression control sequence can comprise a promoter region, and the promoter region can comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein the promoter A portion may be fused to a portion of a promoter of another cellular protein (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not contain additional introns.
  • a promoter of another cellular protein eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • Such vectors may include, for example, plasmids, cosmids, viruses, bacteriophages or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the application provides one or more cells (eg, host cells) or non-human organisms, which can comprise or express an OX40 antigen polypeptide described herein, a fusion protein or immunoassay as described herein A compound, one or more isolated nucleic acid molecules described herein, or one or more vectors described herein.
  • a vector described herein can be introduced into the cell (eg, a host cell), such as a prokaryotic cell (eg, a bacterial cell), a CHO cell, an NS/0 cell, a HEK293T cell, or a HEK293A cell, or other eukaryotic cell.
  • a prokaryotic cell eg, a bacterial cell
  • CHO cell e.g, an NS/0 cell
  • HEK293T cell e.g., HEK293T cell
  • HEK293A cell eukaryotic cell
  • Vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the non-human organism can be a transgenic or genetically engineered organism that can be derived from or comprise the cell (eg, a host cell).
  • the application provides a method of making the OX40 antigen polypeptide or the fusion protein or immunoconjugate.
  • the method can comprise culturing a cell (eg, a host cell) described herein under conditions such that the OX40 antigen polypeptide or the fusion protein or immunoconjugate is expressed.
  • a cell eg, a host cell
  • such methods can be understood by those of ordinary skill in the art by using appropriate media, appropriate temperatures and incubation times, and the like.
  • the method can further comprise the step of isolating and/or purifying the OX40 antigen polypeptide or the fusion protein or immunoconjugate.
  • affinity chromatography can be carried out using protein G-agarose or protein A-agarose, and the OX40 antigen polypeptide described in the present application can be purified and isolated by gel electrophoresis and/or high performance liquid chromatography or the like. Fusion protein or immunoconjugate.
  • compositions and vaccines are provided.
  • the application provides a composition (eg, a pharmaceutical composition), which can comprise an OX40 antigen polypeptide as described herein, a fusion protein or immunoconjugate as described herein, as described herein A nucleic acid molecule, a vector described herein, or a cell (e.g., a host cell) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • a composition eg, a pharmaceutical composition
  • a composition eg, a pharmaceutical composition
  • a composition which can comprise an OX40 antigen polypeptide as described herein, a fusion protein or immunoconjugate as described herein, as described herein
  • a nucleic acid molecule e.g., a vector described herein, or a cell (e.g., a host cell) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or nonionic surfaces. Active agent, etc.
  • the pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques in the art, for example according to Remington: The Science and The technique disclosed in Practice of Pharmacy, Nineteenth Edition, Gennaro, ed., Mack Publishing Co., Easton, PA, 1995.
  • the pharmaceutical composition can be used to produce an immunologically active substance in vivo (e.g., in an organism, such as an animal or in a human body) or in vitro (e.g., in an isolated cell or tissue).
  • the immunologically active substance may comprise an OX40 specific antibody, such as an OX40 agonistic antibody, such as antibody DF004.
  • the pharmaceutical composition is for inhibiting tumor growth (eg, a hematoma or a solid tumor).
  • the pharmaceutical compositions of the present application can inhibit or delay the progression or progression of the disease, can reduce tumor size (even substantially eliminate tumors), and/or can alleviate and/or stabilize disease states.
  • the pharmaceutical composition described herein may be in a form suitable for oral administration, such as a tablet, capsule, pill, powder, sustained release formulation, solution, suspension, or for parenteral injection, such as Sterile solutions, suspensions or emulsions, or for topical administration as an ointment or cream or as a suppository for rectal administration.
  • the pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages.
  • the pharmaceutical composition may further comprise a conventional pharmaceutical carrier or excipient.
  • the pharmaceutical composition may include other drugs or agents, carriers, adjuvants and the like.
  • the pharmaceutical compositions described herein may comprise a therapeutically effective amount of the OX40 antigen polypeptide or the fusion protein or immunoconjugate.
  • the therapeutically effective amount is an amount that is capable of producing an immunologically active substance (eg, an OX40-specific antibody, such as an OX40 agonistic antibody), and/or is capable of treating or slowing the progression or progression of a disease (eg, a tumor).
  • the specific amount/concentration of the dose may vary depending on the method of administration and the needs of the patient, and may be determined based on, for example, patient volume, viscosity, and/or body weight, and the like. It will be appreciated that those skilled in the art (e.g., a physician or pharmacist) can readily adjust these particular dosages based on the particular patient, formulation, and/or condition of the disease.
  • the pharmaceutical composition described herein can be a vaccine (eg, a cancer vaccine).
  • a vaccine which may comprise one or more substances selected from the group consisting of: and optionally an immunogenic carrier: 1) one or more of the OX40 described herein An antigenic polypeptide; 2) a fusion protein or immunoconjugate as described herein; 3) a nucleic acid molecule as described herein; 4) a vector described herein; 5) a cell (eg, a host cell) as described herein And 6) a composition (eg, a pharmaceutical composition) as described herein.
  • the vaccine is a cancer vaccine.
  • Vaccines may also comprise one or more additional antigens (eg, tumor antigens).
  • the additional antigen may comprise one or more additional OX40 antigen polypeptides.
  • the vaccine can be used to treat one or more cancers in a subject (eg, a mammal, such as a human) (eg, a blood cancer or solid tumor, including with T cells, particularly CD4 + and/or CD8 + T Cell-associated cancer).
  • the cancer may include, but is not limited to, for example, melanoma, brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma, gastrointestinal cancer (such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer), liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell carcinoma, prostate cancer, and/or renal cell carcinoma.
  • melanoma brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma
  • gastrointestinal cancer such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer
  • liver cancer such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer
  • liver cancer such as lip cancer, oral cancer, esophageal cancer, small
  • the isolated OX40 antigen polypeptide described herein or the fusion protein or immunoconjugate can be prepared by chemical synthesis or recombinant expression, purification, etc., and the OX40 antigen polypeptide or the fusion protein or immunoconjugate can be subsequently conjugated
  • the vaccine eg, a cancer vaccine
  • the vaccine is prepared by mixing with one or more other tumor antigens and/or pharmaceutically acceptable adjuvants.
  • one or more immunogenic carriers or adjuvants may be included in the vaccine.
  • the OX40 antigen polypeptide, the fusion protein or immunoconjugate or other tumor antigen can be combined or combined with one or more glycolipid analogs to enhance its immune effect.
  • vaccines or compositions can be used to immunize (e.g., by intramuscular or parenteral administration) subjects (e.g., animals or humans, such as rodents, rabbits, sheep, etc.).
  • the vaccine may also comprise one or more pharmaceutically acceptable adjuvants such as buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions , metal complexes and/or nonionic surfactants, and the like.
  • Antibody and its production, screening method and use
  • the application provides an antibody that is capable of specifically binding to an OX40 antigen polypeptide as described herein.
  • the antibody can be an OX40 agonistic antibody.
  • the antibody is a monoclonal antibody.
  • the antibody does not substantially bind to an OX40 (eg, human OA40) variant comprising the mutant G70A and/or the mutant F71A.
  • the antibody may not substantially bind to a human OX40 variant having the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 36.
  • the antibody competes with DF004 for binding to the OX40 antigen polypeptide of the present application. In some cases, the antibody may not be DF004.
  • the antibody DF004 described herein comprises a light chain comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is represented by SEQ ID NO: 63, and the amino acid sequence of the LCDR2 is SEQ ID NO: 64 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:65.
  • the heavy chain comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 66, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 67 and the amino acid sequence of the HCDR3 is SEQ. ID NO: 68 is shown.
  • the heavy chain variable region amino acid sequence of the antibody DF004 described herein is set forth in SEQ ID NO: 70, and the light chain variable region amino acid sequence thereof is set forth in SEQ ID NO: 69.
  • the heavy chain amino acid sequence of the antibody DF004 described herein is set forth in SEQ ID NO: 56, and the light chain amino acid sequence thereof is set forth in SEQ ID NO:58.
  • the application provides a pharmaceutical composition that can comprise an antibody (eg, an OX40 agonistic antibody) as described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or nonionic surfaces. Active agent, etc.
  • the application provides a method for producing an OX40 agonistic antibody.
  • the method can comprise administering (eg, to a subject, such as an animal or human to be immunized; or in vitro, eg, to an isolated cell or tissue) an OX40 antigen polypeptide as described herein or a fusion protein described herein. Or an immunoconjugate.
  • the method can comprise immunizing an animal with an OX40 antigenic polypeptide described herein.
  • the method further comprises isolating an antibody that specifically binds to the OX40 antigen polypeptide.
  • the application provides the use of an OX40 antigen polypeptide described herein or a fusion protein or immunoconjugate described herein for the preparation of an immunologically active substance, which can comprise an OX40 agonistic antibody.
  • the OX40 agonistic antibodies produced can be used to treat diseases (eg, cancers, such as blood cancers or solid tumors, particularly cancers associated with the expression or regulation of OX40).
  • diseases eg, cancers, such as blood cancers or solid tumors, particularly cancers associated with the expression or regulation of OX40.
  • the application provides the use of the OX40 antigen polypeptide or the fusion protein or immunoconjugate for the preparation of a medicament for treating a disease (eg, a cancer, such as a blood cancer) Or a solid tumor, especially a cancer associated with the expression or regulation of OX40).
  • the cancer may include, but is not limited to, for example, melanoma, brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma, gastrointestinal cancer (such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer), liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell carcinoma, prostate cancer, and/or renal cell carcinoma.
  • melanoma brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma
  • gastrointestinal cancer such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer
  • liver cancer such as lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer
  • liver cancer such as lip cancer, oral cancer, esophageal cancer, small
  • Antibodies that can be produced, prepared, screened, and/or detected using the OX40 antigen polypeptides described herein or the fusion proteins or immunoconjugates described herein can include naturally occurring antibodies as well as antibodies produced by recombinant methods. These antibodies can include polyclonal and monoclonal antibodies prepared using techniques known in the art. These antibodies may be OX40 agonistic antibodies, for example, which are capable of activating OX40 or enhancing its activity. The antibody can be used to treat and/or prevent a disease associated with the expression or activity of OX40 and/or OX40L, such as a tumor (such as a blood cancer or a solid tumor). The antibodies can also be used in immunoassays, for example, to characterize, identify or label tumor tissues and/or other tissues or cells in the tumor microenvironment, such as immune cells, such as immune cells expressing OX40.
  • an OX40 antigen polypeptide described herein or a fusion protein or immunoconjugate described herein can be injected or otherwise administered to a host animal (including, but not limited to, rabbits, mice, rats, etc.) To induce the production of serum containing polyclonal antibodies specific for the OX40 antigen, fusion protein or immunoconjugate.
  • OX40 antigen, fusion protein or immunoconjugate can be carried out by one or more injections, and an immunological adjuvant can also be administered as needed.
  • various adjuvants can be used to increase the immune response, including but not limited to, Freund's reagent (complete or incomplete), inorganic adjuvants (such as aluminum hydroxide), surface active substances (such as lysolecithin) , complex polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenols, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.
  • adjuvants examples include MPL-TDM adjuvant (monophosphate lipid A, synthetic trehalose dimycolate).
  • MPL-TDM adjuvant monophosphate lipid A, synthetic trehalose dimycolate.
  • Specific protocols for immunization are known to those skilled in the art and can be carried out by any means that induces an immune response in a selected animal host.
  • the immunogen (with or without an adjuvant) can be injected into the host animal by multiple subcutaneous or intraperitoneal injections, or intramuscularly or by intravenous infusion.
  • the immunogen may comprise an isolated OX40 antigen polypeptide of the present application, a fusion protein or immunoconjugate of the present application, and the like.
  • the immunogen eg, an isolated OX40 antigen polypeptide of the present application
  • the immunogenic protein is conjugated in the host animal to be immunized.
  • the conjugation can be achieved by chemical coupling, fusion protein based methods, or other methods known to those skilled in the art.
  • the immunogenic protein in the host animal to be immunized may include, for example, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovine thyroprotein, soybean trypsin inhibitor, and promiscuous T helper peptide.
  • the antibody produced or prepared is a monoclonal antibody
  • methods known to those skilled in the art such as hybridoma technology can be employed (see U.S. Patent 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory). Prepared by Press, Second Edition (1988) and Hammerling et al, Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), recombinant DNA methods or other methods known to those skilled in the art.
  • Other examples of methods that can be used to prepare monoclonal antibodies include, but are not limited to, human B-cell hybridoma technology, EBV-hybridoma technology, and the like.
  • the monoclonal antibodies produced can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subtype thereof.
  • Hybridomas useful for the production of monoclonal antibodies can be cultured in vitro or in vivo.
  • a host eg, a mouse, a humanized mouse, a mouse with a human immune system, a hamster, a rabbit, a camel, or any other suitable host animal
  • a host eg, a mouse, a humanized mouse, a mouse with a human immune system, a hamster, a rabbit, a camel, or any other suitable host animal
  • an antibody capable of specifically binding to a protein for immunization such as an isolated OX40 antigen polypeptide of the present application, or a fusion protein or immunoconjugate of the present application, can be generated.
  • lymphocytes can also be immunized in vitro, and then the lymphocytes can be fused with myeloma cells by using a suitable fusing agent (e.g., polyethylene glycol) to form a hybridoma.
  • a suitable fusing agent e.g., polyethylene glycol
  • peripheral blood lymphocytes can be used in the preparation of hybridomas for the production of antibodies, such as human cells.
  • spleen cells or lymph node cells can be used.
  • the lymphocytes can then be fused with an immortalized cell line by the use of a suitable fusing agent, such as polyethylene glycol, to form a hybridoma.
  • the immortalized cell line is typically a transformed mammalian cell, particularly a rodent, bovine or human myeloma cell.
  • a myeloma cell line of rat or mouse can be used.
  • the hybridoma cells can be cultured in a suitable medium.
  • the medium can comprise one or more substances that inhibit the growth or survival of unfused, immortalized cells.
  • the medium used for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine ("HAT medium"). They are substances that prevent the growth of HGPRT-deficient cells.
  • the immortalized cell line used may be a cell line capable of efficiently fusing, supporting stable production of selected antibody-producing cells, producing antibodies at a high level, and being sensitive to a medium such as HAT medium.
  • myeloma cell lines may include murine myeloma cell lines such as MOPC-21 and MPC-11 mouse tumor cell lines, cell lines derived from SP2/0, and the like.
  • Antibodies can also be prepared using human myeloma, murine-human heteromyeloma cell line, mouse hybridoma cell line NSO, and the like.
  • the produced monoclonal antibody and the isolated OX40 antigen polypeptide, the fusion protein or immunoconjugate can be detected by an in vitro binding assay such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or the like. Binding affinity and/or binding specificity. These techniques are within the skill of those in the art.
  • the clone can be subcloned by limiting dilution and cultured in a standard manner.
  • Suitable media can include, for example, DMEM or RPMI-1640 medium.
  • the cell culture can then be removed from the cell culture using conventional immunoglobulin purification techniques (eg, Protein A-Sepharose, Hydroxyapatite Chromatography, Gel Exclusion Chromatography, Gel Electrophoresis, Dialysis, or Affinity Chromatography).
  • immunoglobulin purification techniques eg, Protein A-Sepharose, Hydroxyapatite Chromatography, Gel Exclusion Chromatography, Gel Electrophoresis, Dialysis, or Affinity Chromatography.
  • the monoclonal antibody secreted by the subclone is isolated from the host animal ascites or serum.
  • the monoclonal antibody can also be produced or produced by other methods, such as recombinant DNA methods.
  • DNA isolated from hybridomas can be inserted into an expression vector and subsequently transfected into host cells (eg, E. coli cells, NSO cells, ⁇ COS cells, Chinese hamster ovary (CHO) cells, or otherwise no immunity generated)
  • host cells eg, E. coli cells, NSO cells, ⁇ COS cells, Chinese hamster ovary (CHO) cells, or otherwise no immunity generated
  • the globulin myeloma cells thereby synthesizing the monoclonal antibody in the recombinant host cell.
  • the application provides a method of preventing the formation of OX40-OX40L hexamer, the method comprising administering an agent that specifically binds to an OX40 antigen polypeptide described herein.
  • OX40 and OX40L are unable to recombine after administration of an agent that specifically binds to the OX40 antigen polypeptide described herein in vivo or in vitro.
  • OX40 and OX40L are still capable of binding to each other after in vivo or in vitro administration of an agent that specifically binds to the OX40 antigen polypeptide described herein, but are unable to form a hexamer (eg, only a dimer) , 3-mer, 4-mer, etc.). In some cases, OX40 and OX40L cannot bind to each other or bind to an active molecular conformation.
  • an agent that specifically binds to an OX40 antigen polypeptide described herein can be an agonist of OX40.
  • the agent that specifically binds to an OX40 antigen polypeptide described herein is an OX40 agonistic antibody (eg, antibody DF004 described herein).
  • the application provides the use of the OX40 antigen polypeptide or the fusion protein or immunoconjugate for the preparation of a reagent useful for preventing the formation of OX40-OX40L hexamer.
  • the agent is capable of activating OX40 as an agonist.
  • the agent can include or can be an OX40 agonistic antibody (eg, antibody DF004 as described herein).
  • the application provides methods for screening and/or detecting OX40 agonistic antibodies.
  • the method can comprise allowing a candidate antibody or antigen-binding fragment thereof to be screened and/or to be detected, or a cell or cell secretion expressing the candidate antibody or antigen-binding fragment thereof, and an OX40 antigen polypeptide or present described herein.
  • the fusion protein or immunoconjugate described in the application is contacted.
  • the method can also include determining whether the candidate antibody or antigen-binding fragment thereof specifically binds to the OX40 antigen polypeptide, and when the specific binding is detected, identifying the candidate antibody as an OX40 agonistic antibody.
  • the OX40 agonistic antibody can include DF004.
  • the application provides the use of the OX40 antigen polypeptide or a fusion protein or immunoconjugate described herein for the preparation of a reagent useful for detecting, identifying and/or screening OX40 agonistic antibodies .
  • the OX40 agonistic antibody can comprise DF004.
  • a cell (cell secretion), a cell culture mixture, or a substance (eg, an agent or a mixture) comprising an antibody or antigen-binding fragment thereof to be screened/detected can be made to the present application by using the cell to be detected, identified, and/or screened.
  • a substance eg, an agent or a mixture
  • an antibody or antigen-binding fragment thereof to be screened/detected can be made to the present application by using the cell to be detected, identified, and/or screened.
  • the isolated OX40 antigen polypeptide is contacted or contacted with a fusion protein or immunoconjugate as described herein, and then, depending on whether it is capable of specifically binding to an isolated OX40 antigen polypeptide as described herein (or as described herein)
  • the OX40 antigen polypeptide in the fusion protein or immunoconjugate is used to determine whether the cell, cell secretion, cell culture mixture or the reagent or mixture contains an OX40 agonistic antibody or antigen-binding fragment thereof. In certain embodiments, whether the cell, cell secretion, cell culture mixture, or the reagent or mixture contains antibody DF004 or an antigen-binding fragment thereof can be determined by this method.
  • the screening and/or detection can be performed by, for example, an in vitro binding assay such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • an in vitro binding assay such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • the application provides methods of preventing and/or treating cancer in a subject.
  • the method can comprise administering to the subject an effective amount of one or more substances selected from the group consisting of: 1) an OX40 antigen polypeptide as described herein; 2) a fusion protein or immunization described herein a conjugate; 3) a nucleic acid molecule as described herein; 4) a vector as described herein; 5) a cell (eg, a host cell) as described herein; 6) a pharmaceutical composition as described herein;
  • the vaccine described herein may be, for example, a blood cancer or a solid tumor, particularly a cancer associated with the expression or regulation of OX40.
  • the cancer may include, but is not limited to, melanoma, brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma, gastrointestinal cancer (eg, lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer), liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell carcinoma, prostate cancer, and/or renal cell carcinoma.
  • melanoma brain cancer, bone cancer, leukemia, lymphoma, epithelial cell carcinoma, adenocarcinoma
  • gastrointestinal cancer eg, lip cancer, oral cancer, esophageal cancer, small intestine cancer, Gastric cancer, colon cancer
  • liver cancer eg, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell carcinoma, prostate cancer, and/or renal cell carcinoma.
  • the full-length amino acid sequences of the heavy chain and the light chain of the anti-OX40 activating antibody DF004 were obtained, and the corresponding DNA sequence was designed by the DNAworks online tool (http://helixweb.nih.gov/dnaworks/) and passed.
  • the light chain gene of DF004 was obtained by a synthetic method and designated as DF004-LC (the nucleotide sequence is shown in SEQ ID NO: 57).
  • the heavy chain gene of DF004 was obtained in the same manner and designated as DF004-HC (the nucleotide sequence is shown as SEQ ID NO: 55).
  • the synthesized DF004-HC and DF004-LC were double-digested by Thermo's HindIII and PmeI into the commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20), and the accuracy of the constructed plasmid was verified by sequencing.
  • the recombinant plasmid DNAs are: pcDNA4-DF004HC and pcDNA4-DF004LC.
  • the two recombinant plasmids pcDNA4-DF004HC and pcDNA4-DF004LC obtained above were diluted with 6 mL of Freestyle 293 medium and added to a PEI (Polyethylenimine) solution for transformation.
  • PEI Polyethylenimine
  • Each set of plasmid/PEI mixture was separately added to a 600 mL cell suspension, placed at 37 ° C, 10% CO 2 , 90 rpm; and 50 ⁇ g/L IGF-1 was added.
  • 600 mL of EX293 medium, 2 mM glutamine and 50 ⁇ g/L IGF-1, 135 rpm were added. Add 3.8 mM VPA after 24 hours.
  • DF004 antibody transient expression culture supernatant After 5 to 6 days of culture, 1200 mL of the DF004 antibody transient expression culture supernatant was collected, and the DF004 antibody sample was initially purified by ProteinA affinity chromatography. The obtained protein sample was subjected to preliminary detection by SDS-PAGE, and the target band was clearly seen.
  • the amino acid sequence of the human OX40 extracellular domain was obtained from the uniport database (P43489, position 1 to position 170, the amino acid sequence of which is shown in SEQ ID NO: 43), and the DNAworks online tool (http://helixweb.nih) was used. .gov/dnaworks/) Designed the corresponding coding DNA sequence and obtained a nucleic acid molecule encoding the OX40 extracellular domain by artificial synthesis, named OX40.
  • the synthesized OX40 nucleic acid molecule was double-enzyme-purified by Thermo's HindIII and AgeI into the commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20), and the accuracy of the constructed plasmid was verified by sequencing, and the recombinant plasmid DNA was obtained: pcDNA4-OX40-His (wherein the nucleotide sequence of OX40-His is shown as SEQ ID NO: 60).
  • telomere sequence SEQ ID NO: 71, SEQ ID NO, respectively
  • pcDNA4-DF004VH-CH1-His The plasmid DNA is: pcDNA4-DF004VH-CH1-His (wherein the nucleotide sequence of DF004VH-CH1-His is as shown in SEQ ID NO: 62).
  • the recombinant expression vectors obtained in the above examples were divided into two groups, group 1 was 200 ⁇ g pcDNA4-OX40-His; group 2 was 100 ug pcDNA4-DF004-VH-CH1-His+100 ⁇ g pcDNA4-DF004LC.
  • Each set of plasmids was diluted with 6 mL of Freestyle 293 medium and a PEI (Polyethylenimine) solution required for transformation was added.
  • Each set of plasmid/PEI mixture was separately added to a 600 mL cell suspension, placed at 37 ° C, 10% CO 2 , 90 rpm; and 50 ⁇ g/L IGF-1 was added.
  • the crystal conditions were optimized as follows: 0.1 M potassium tartrate, 10% to 30% PEG 3350. The optimal result was 0.1 M sodium potassium tartrate and 14% PEG 3350. Further based on the above conditions, glycerol was added, and the protocol was as follows: 0.1 M potassium tartrate, 14% PEG 3350, 5% to 25% glycerol. The optimum conditions were 0.1 M potassium tartrate, 14% PEG 3350, 10% glycerol, and the complex was crystallized. The obtained crystal was subjected to X-ray diffraction, and the obtained diffraction data was processed by CCP4 software, and then a schematic diagram was made using PyMol software, and the results are shown in Fig. 2.
  • point mutations were performed on key sites by overlapping PCR.
  • the key mutation strategies were: W86A, R65A, P83A, A126V, G70A and F71A.
  • the primers used for point mutations are shown in the table below.
  • the overlapping PCR products were double-digested with Thermo-type HindIII and AgeI into the commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) to obtain recombinant plasmid DNA: pcDNA4-OX40W86A, pcDNA4-OX40R65A, pcDNA4-OX40P83A, pcDNA4-OX40A126V, pcDNA4-OX40G70A and pcDNA4-OX40F71A.
  • the accuracy of the above recombinant plasmid constructed was verified by sequencing.
  • the 6 recombinant expression vectors obtained in Example 3.1 were diluted with 6 mL of Freestyle 293 medium and added to the PEI (Polyethylenimine) solution required for transformation.
  • PEI Polyethylenimine
  • Each set of plasmid/PEI mixture was separately added to a 600 mL cell suspension, placed at 37 ° C, 10% CO 2 , 90 rpm; and 50 ⁇ g/L IGF-1 was added.
  • 600 mL of EX293 medium, 2 mM Glutamine and 50 ug/L IGF-1, 135 rpm were added. Add 3.8 mM VPA after 24 hours.
  • OX40 antigen transient expression culture supernatant After 5 to 6 days of culture, 6 ⁇ 1200 mL of OX40 antigen transient expression culture supernatant was collected, and the antigen after OX40 point mutation was purified by Ni-NTA affinity chromatography, and the target band was clearly observed.
  • OX40-His W86A OX40-His R65A
  • OX40-His P83A OX40-His G70A
  • OX40-His F71A OX40-His A126V
  • SEQ ID NO: 87 amino acid sequence selected from the group consisting of SEQ ID NO: : 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 90.
  • OX40-His Diluted the above 6 mutant antigens of OX40-His with coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6) (OX40-His W86A, OX40-His R65A, OX40-His P83A, OX40-His G70A, OX40 -His F71A and OX40-His A126V) and wild-type antigen OX40-His, diluted to a concentration of 5 ⁇ g/ml, 100 ⁇ l/well, overnight at 4 ° C; after washing, 3% BSA-PBS was blocked at 37 ° C for 2 hours.
  • coating buffer 50 mM Na 2 CO 3 , NaHCO 3 pH 9.6
  • the DF004 antibody was started from 10 ⁇ g/ml, diluted 3 times, a total of 11 concentrations, and the dilution (1% BSA-PBS) was used as a control, and incubated at 37 ° C for 2 hours; after washing, goat anti-human IgG-HRP was added ( Goat anti-human IgG-HRP conjugated, Sigma: A0170), incubated for 1 hour at 37 °C. After washing the plate, a soluble one-component TMB substrate coloring solution was added, and the color was developed at room temperature for 5-10 minutes. The color reaction was terminated by adding 2N H 2 SO 4 50 ⁇ L/well.
  • the OD 450nm-650nm value was read on the MD SpectraMax Plus 384 microplate reader, and the software software SoftMax Pro v5.4 was used for data processing and mapping analysis.
  • the results are shown in Fig. 4. It can be seen from the results that the mutation of F71A directly causes OX40 to not bind to DF004, and the G70A mutation causes the affinity of OX40 to bind DF004 to be greatly reduced, and the other mutations do not substantially inhibit the binding of OX40 to DF004.
  • the DF004 antibody was diluted to 10 ug/ml with PBST (PBS containing 0.05% Tween 20) and each OX40-His antigen variant was diluted to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM. 200 ⁇ l of PBST, DF004 antibody dilution, different concentrations of antigen dilution and 10 mM pH 1.7 Gly-HCl were added to a 96-well plate (Greinerbio-one, cat #655209) and placed in an Octet K2 card slot.
  • PBST PBS containing 0.05% Tween 20
  • AHC fortebio, cat#18-5060
  • AHC fortebio, cat#18-5060
  • Data analysis and kinetic curve fitting were performed using Data Analysis 9.0 software, and the results are shown in Figures 5 and 6A-6G.
  • Figures 6A-6G show, in turn, curve fitting of variants OX40-His A126V, OX40-His P83A, OX40-His R65A, OX40-His W86A, OX40-His F71A, OX40-His G70A, and control wild-type OX40-His result. It can be seen from the results that the affinity of the variant OX40-His G70A and DF004 is greatly decreased, and the mutation of the variant OX40-His F71A results in no binding to DF004, and the other variants do not destroy the binding to DF004.

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Abstract

提供一种分离的OX40抗原多肽，可用于制备特异性结合OX40的抗体，和/或能够抑制OX40配体与OX40相结合的抗体。

Description

OX40抗原多肽及其用途 技术领域
本申请涉及抗体领域，特别是用于制备特异性抗体的抗原多肽。具体而言，本申请涉及分离的OX40抗原多肽，以及其用于产生、识别或筛选免疫活性物质(例如抗体)的用途。
背景技术
T细胞活化需要两种信号协同作用：第一信号由T细胞抗原受体(TCR)识别抗原产生，经CD3分子将信号转导至细胞内，第一信号决定T细胞在适应性免疫应答中的特异性；第二信号是由抗原提呈细胞(APC)或者靶细胞表面的共刺激分子与T细胞表面的相应的协同刺激分子受体相互作用而产生。共刺激信号刺激抗原特异性T细胞增殖，并分化为效应T细胞。如果缺乏共刺激信号，T细胞则会进入无反应或自身免疫耐受状态，甚至进入程序性死亡。
OX40是TNFR受体超家族成员，是I型跨膜糖蛋白，OX40主要表达于活化的CD4 +T细胞和CD8 +T细胞上(Paterson等(1987)MolImmunol24:1281–1290)。OX40的胞外区由3个半胱氨酸富集域(CRD)和一个C端不完全CRD组成(DeanneM等(2006)Structure14:1321-1330)。OX40是次级共刺激分子。与CD28不同，OX40不表达于静息的T细胞表面，而在T细胞活化24-72小时有较高的表达。OX40的配体OX40L(TNFSF4，TXGP1，OX-40L，gp34或CD252)是II型跨膜糖蛋白，表达于活化的抗原提呈细胞，如树突状细胞、B细胞等(Godfrey,W.R等(1994)JExpMed180:757–762)。OX40/OX40L信号在T细胞的活化、增殖以及抑制凋亡过程中发挥着非常重要的作用。
研究表明激活型的OX40抗体能够有效地促进T细胞增殖和活化，并产生较好的抗肿瘤作用(Brendan D.Curti等(2013)CancerRes73:7189-7198)。
然而，人们对于激活型OX40抗体的作用位点及能够用于筛选和产生激活型OX40抗体的抗原表位仍了解甚少。因此，缺乏适当的手段来识别和筛选激活型OX40抗体，或者产生激活型OX40抗体，进而用于激活免疫系统或治疗疾病。
发明内容
本申请提供了一种分离的OX40抗原多肽。本申请的OX40抗原多肽能够特异性结合OX40激动性抗体，并且可用于产生所述OX40激动性抗体。所述OX40激动性抗体能够阻止OX40-OX40L六聚体的形成。本申请的OX40抗原多肽能够用于检测、识别和/或筛选OX40激动性抗体。在某些情形中，本申请的OX40抗原多肽还能够用于制备疫苗，例如癌症疫苗。因此，在某些情形中，本申请的OX40抗原多肽能够用于预防和/或治疗癌症。
一方面，本申请提供了一种分离的OX40抗原多肽，其可包含SEQ ID NO:1-32以及它们的片段或 变体中的任一项或多项所示的氨基酸序列。
所述氨基酸序列的变体可包含：1)与SEQ ID NO:1-32中任一项所示的氨基酸序列具有至少80％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同源性的氨基酸序列；和/或2)在SEQ ID NO:1-32中任一项所示的氨基酸序列中经过取代、缺失或添加一个或几个(例如，1-2个，1-3个，1-4个，1-5个，1-6个，1-7个，1-8个，1-9个，1-10个，1-11个，1-12个或更多)氨基酸而获得的氨基酸序列。
在某些实施方式中，所述氨基酸序列的变体不包含突变G70A和/或突变F71A。在某些实施方式中，所述氨基酸序列的变体不包含残基G70和/或残基F71处的氨基酸突变。在本申请中，残基G70是指相应于SEQ ID NO:33所示氨基酸序列中第70位的残基G，残基F71是指相应于SEQ ID NO:33所示氨基酸序列中第71位的残基F。突变G70A是指将相应于SEQ ID NO:33所示氨基酸序列中第70位的残基G突变为A。突变F71A是指将相应于SEQ ID NO:33所示氨基酸序列中第71位的残基F突变为A。
在本申请中，“残基Xn”是指相应于SEQ ID NO:33所示氨基酸序列中第n位的残基X，其中n为正整数，X为任意氨基酸残基的缩写。
在本申请中，突变“XnY”是指将相应于SEQ ID NO:33所示氨基酸序列中第n位的残基X突变为Y，其中n为正整数，X和Y分别独立地为任意氨基酸残基的缩写，且X不同于Y。
在本申请中，某氨基酸序列中的某残基“相应于”另一氨基酸序列中的某残基通常是指，在优化条件下进行氨基酸序列比对时所获得的残基对应关系。所述序列比对可通过本领域技术人员了解的方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。例如，SEQ ID NO:2所示氨基酸序列中第1位至第7位的残基分别相应于SEQ ID NO:33所示氨基酸序列中第65位至第71位的残基。
在某些实施方式中，本申请所述的分离的OX40抗原多肽包含约4-160个(例如，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，至少15个，至少20个，至少25个，至少30个，至少35个，至少40个，至少45个，至少50个，至少55个，至少60个，至少65个，至少70个，至少75个，至少80个，至少85个，至少90个，至少95个，至少100个，至少110个，至少120个，至少130个，至少140个，至少150个，至少160个或更多个)氨基酸残基。
在某些实施方式中，本申请所述的分离的OX40抗原多肽源自OX40的细胞外结构域，其片段或变体。例如，其可源自人、食蟹猴、小鼠或大鼠OX40的细胞外结构域，其片段或变体。
在某些实施方式中，本申请所述的分离的OX40抗原多肽源自人OX40。例如，其可源自人OX40的 细胞外结构域，其片段或变体。所述人OX40细胞外结构域的氨基酸序列可如SEQ ID NO:43所示。
在某些实施方式中，本申请的分离的OX40抗原多肽源自OX40(例如，人OX40)细胞外结构域中包含下组中一个或多个结构域或其片段的区域：半胱氨酸富集域1(CRD1)、半胱氨酸富集域2(CRD2)和半胱氨酸富集域3(CRD3)。在某些实施方式中，本申请的分离的OX40抗原多肽源自OX40(例如，人OX40)细胞外结构域中包含下组中一个或多个结构域或其片段的区域：半胱氨酸富集域1(CRD1)和半胱氨酸富集域2(CRD2)。在某些实施方式中，本申请的分离的OX40抗原多肽不包含OX40(例如，人OX40)细胞外结构域中的下列区域：半胱氨酸富集域3(CRD3)或半胱氨酸富集域4(CRD4)。
例如，本申请的分离的OX40抗原多肽可包含源自下组中的一个或多个结构域或其片段的区域：1)OX40(例如，人OX40)的CRD1的模块A1；2)OX40(例如，人OX40)的CRD1的模块B2；3)OX40(例如，人OX40)的CRD2的模块A1；和4)OX40(例如，人OX40)的CRD2的模块B2。
例如，人OX40的CRD1中模块A1的氨基酸序列可如SEQ ID NO:45所示，人OX40的CRD1中模块B2的氨基酸序列可如SEQ ID NO:46所示，人OX40的CRD1的氨基酸序列可如SEQ ID NO:47所示。人OX40的CRD2中模块A1的氨基酸序列可如SEQ ID NO:48所示，人OX40的CRD2中模块B2的氨基酸序列可如SEQ ID NO:49所示，人OX40的CRD2的氨基酸序列可如SEQ ID NO:50所示。
此外，人OX40的CRD3的氨基酸序列可如SEQ ID NO:51所示。人OX40的CRD4的氨基酸序列可如SEQ ID NO:52所示。
在某些实施方式中，本申请的分离的OX40抗原多肽具有免疫原性。
另一方面，本申请提供了一种融合蛋白或免疫缀合物，其包含本申请所述的分离的OX40抗原多肽。
另一方面，本申请提供了一种或多种分离的核酸分子，所述核酸分子可编码本申请所述的分离的OX40抗原多肽和/或本申请所述的融合蛋白或免疫缀合物。
在另一个方面，本申请提供了一种或多种载体，所述载体可包含本申请所述的一种或多种分离的核酸分子。
在另一个方面，本申请提供了一种或多种细胞(例如，宿主细胞)或非人生物体，其可包含或表达本申请所述的OX40抗原多肽、本申请所述的融合蛋白或免疫缀合物、本申请所述的一种或多种分离的核酸分子、和/或本申请所述的一种或多种载体。
在另一个方面，本申请提供了一种组合物(例如药物组合物)，其可包含本申请所述的OX40抗原多肽、本申请所述的融合蛋白或免疫缀合物、本申请所述的核酸分子、本申请所述的载体、或本申请所述的细胞(例如，宿主细胞)，以及任选地药学上可接受的佐剂。
在另一个方面，本申请提供了一种疫苗，其可包含一种或多种选自下列的物质以及任选地免疫原性载体：1)一种或多种本申请所述的OX40抗原多肽；2)本申请所述的融合蛋白或免疫缀合物；3)本申请所述的核酸分子；4)本申请所述的载体；5)本申请所述的细胞(例如，宿主细胞)；和6)本申请所述的组合物(例如，药物组合物)。在某些实施方式中，所述疫苗为癌症疫苗。
在另一个方面，本申请提供了一种抗体，其能够特异性结合本申请所述的OX40抗原多肽。例如，所述抗体可以为OX40激动性抗体。在某些实施方式中，所述抗体为单克隆抗体。在某些实施方式中，所述抗体不包括DF004。在某些实施方式中，所述抗体基本上不结合包含突变G70A和/或突变F71A的OX40(例如，人OA40)变体。例如，所述抗体基本上不结合氨基酸序列如SEQ ID NO:35或SEQ ID NO:36所示的人OX40变体。
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗体(例如，OX40激动性抗体)，以及任选地药学上可接受的佐剂。
在另一个方面，本申请提供了一种用于产生OX40激动性抗体的方法。所述方法可以包括用本申请所述的OX40抗原多肽免疫动物。在某些实施方式中，所述方法还可包括分离与所述OX40抗原多肽特异性结合的抗体。
在另一个方面，本申请提供了用于筛选和/或检测OX40激动性抗体的方法。所述方法可包括使待筛选和/或待检测的候选抗体或其抗原结合片段、或者表达所述候选抗体或其抗原结合片段的细胞或细胞分泌物与本申请所述的OX40抗原多肽或本申请所述的融合蛋白或免疫缀合物接触。所述方法还可以包括判断所述候选抗体或其抗原结合片段是否与所述OX40抗原多肽特异性结合，且当检测到所述特异性结合时，将该候选抗体鉴别为OX40激动性抗体。例如，所述OX40激动性抗体可包括DF004。
在另一个方面，本申请提供了本申请所述的OX40抗原多肽或本申请所述的融合蛋白或免疫缀合物用于制备免疫活性物质的用途，所述免疫活性物质可包括OX40激动性抗体。
在另一个方面，本申请提供了本申请所述的OX40抗原多肽、本申请所述的融合蛋白或免疫缀合物或者本申请所述的抗体用于制备药物的用途，所述药物可用于预防和/或治疗癌症。
在另一个方面，本申请提供了一种阻止OX40-OX40L六聚体形成的方法，所述方法包括施用与本申请所述的OX40抗原多肽特异性结合的试剂。例如，与本申请所述的OX40抗原多肽特异性结合的试剂可以为OX40的激动剂。在某些实施方式中，与本申请所述的OX40抗原多肽特异性结合的试剂为OX40激动性抗体。
在另一个方面，本申请提供了所述OX40抗原多肽或本申请所述的融合蛋白或免疫缀合物用于制备试剂的用途，所述试剂可用于阻止OX40-OX40L六聚体的形成。在某些实施方式中，所述试剂能够作为激 动剂激活OX40。例如，所述试剂可以为OX40激动性抗体。
在另一个方面，本申请提供了所述OX40抗原多肽或本申请所述的融合蛋白或免疫缀合物用于制备试剂的用途，所述试剂可用于检测、识别和/或筛选OX40激动性抗体。在某些实施方式中，所述OX40激动性抗体可包括DF004。
在另一个方面，本申请提供了预防和/或治疗受试者中的癌症的方法。所述方法可包括向所述受试者施用有效量的、选自下组的一种或多种物质：1)本申请所述的OX40抗原多肽；2)本申请所述的融合蛋白或免疫缀合物；3)本申请所述的核酸分子；4)本申请所述的载体；5)本申请所述的细胞(例如宿主细胞)；6)本申请所述的药物组合物；和7)本申请所述的疫苗。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
图1显示了不同物种OX40片段的氨基酸序列比对结果。
图2显示了OX40与其配体OX40L形成的复合物，以及OX40与其激动性抗体形成的复合物。
图3显示了与OX40激动性抗体特异性结合的OX40表位序列，其中框内标示的是所述结合涉及的主要氨基酸残基。
图4显示了OX40表位多肽中的氨基酸序列突变对其与抗体的结合的影响。
图5显示了不同位置突变的OX40表位多肽与抗体结合的动力学参数检测结果。
图6A-6G显示了不同位置突变的OX40表位多肽与抗体结合的动力学曲线拟合结果。
具体实施方式
以下由特定的具体实例说明本发明的实施方式，本领域技术人员可通过本说明书所公开的内容了解本发明的其他优点及功效。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子或片段。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单链抗体(例如,scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及 本文所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CRD1、FR2、CRD2、FR3、CRD3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，术语“抗原结合部分”通常是指抗体中发挥特异性结合抗原功能的一个或多个片段。抗体的抗原结合功能可通过抗体的全长片段来实现。抗体的抗原结合功能也可通过以下的片段来实现：(1)Fab片段，即由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(3)由VH和CH结构域组成的Fd片段；(4)由抗体单臂的VL和VH结构域组成的Fv片段；(5)由VH结构域组成的dAb片段(Ward等，(1989)Nature 341：544-546)；(6)分离的互补决定区(CDR)和(7)可任选地通过连接子连接的两个或两个以上分离的CDR的组合。此外，“抗原结合部分”还可包括由VL和VH配对形成的一价单链分子Fv(scFv)(参见Bird等(1988)Science 242：423-426；以及Huston等(1988)Proc.Natl.Acad.Sci.85：5879-5883)。所述“抗原结合部分”还可包括免疫球蛋白融合蛋白，所述融合蛋白包含选自以下的结合结构域：(1)与免疫球蛋白铰链区多肽融合的结合结构域多肽；(2)与铰链区融合的免疫球蛋白重链CH2恒定区；和(3)与CH2恒定区融合的免疫球蛋白重链CH3恒定区。
在本申请中，术语“OX40”通常是指OX40蛋白，可包括天然地或经遗传改造后含有OX40蛋白基因的细胞所表达的任何OX40蛋白及其变体、功能性片段、同工型(isoform)和种间同源物(species homologs)。OX40是一种I型跨膜糖蛋白，它通过与其配体OX40L相互作用形成多聚体(例如，六聚体)而发挥作用。例如，术语“OX40”可包括与UniProt登记号P43489.1具有至少80％氨基酸序列同一性、并且特异性结合OX40L的多肽或其片段。本申请中的术语“OX40”包括人OX40(hOX40)，hOX40的变体、同工型和种间同源物，以及与hOX40具有至少一个共同表位的其类似物。例如，本申请的OX40蛋白可为人OX40蛋白。人OX40蛋白可包含如SEQ ID NO：xxx所示的氨基酸序列或该氨基酸序列的变体，例如，与其具有至少70％，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％或至少100％序列同源性的变体。
在本申请中，术语“单克隆抗体”通常是指由单一B细胞克隆产生的高度均一、仅针对某一特定抗原表位的抗体。单克隆抗体显示针对一种抗原表位的特异性和亲和性。例如，单克隆抗体可通过杂交瘤产生，所述杂交瘤可以为将具有分泌特异性抗体能力的B细胞和具有无限繁殖能力的骨髓瘤细胞融合后得到的 杂交瘤细胞。
在本申请中，术语“表位”通常是指抗原决定簇，即指分子中被免疫系统识别(例如被抗体识别)的部分。例如，表位可以是被免疫系统识别的抗原上不连续的三维位点。表位通常由分子的化学活性表面基团(例如氨基酸或糖侧链)组成，并通常具有特定的三维结构特征以及特定的电荷特征。表位根据结构，可分为构象表位和非构象表位(线性表位)。构象表位与非构象表位的区别在于在变性溶剂存在下前者丧失结合，而后者则不会。只位于抗原物质表面、易与抗原识别受体或抗体结合的表位可称为功能性表位；位于分子内部、无免疫原性的表位可称为隐藏性表位。
在本申请中，术语“胞外结构域”通常是指蛋白质的细胞外结构域(extracellular domain)。例如，OX40蛋白的胞外结构域通常可为基本不含跨膜和胞质结构域的OX40多肽形式。其中，跨膜的界定是根据本领域中用于鉴定疏水结构域类型的常规标准所鉴定的。跨膜的结构域的确切边界可有所不同，但是大部分是相似的。例如，所述OX40蛋白的胞外结构域可在跨膜结构域或细胞外结构域边界的任一侧含有约5个(或4个、3个、2个或1个)氨基酸。例如，所述OX40蛋白的胞外结构域可以带有相关的信号肽。
在本申请中，术语“分离的核酸分子”或“分离的核酸”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“宿主细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗原多肽的个体细胞，细胞系或细胞培养物。所述宿主细胞可以包括单个宿主细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗原多肽即可。所述宿主细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述宿主细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方案中，所述宿主细胞是哺乳动物细胞。在本申请中，术语“重组宿主细胞”通常指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“肿瘤”通常是指哺乳动物中的机体(例如，细胞或其组成部分)在各种致瘤因子作用下，局部组织细胞增生所形成的赘生物。在本申请中，肿瘤可以包括淋巴瘤、母细胞瘤、肉瘤和血液肿 瘤等。
在本申请中，术语“药学上可接受的佐剂”通常是指任何与所施用的活性成分相容的溶剂、分散介质、涂层、等渗剂和吸收延迟剂等佐剂、赋形剂或其它药物载体。
在本申请中，术语“受试者”通常是指任何人或非人动物。术语“非人动物”可包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、山羊、绵羊、狗、牛、鸡、两栖动物、爬行动物等。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本申请中，术语“OX40抗原多肽”、“OX40表位多肽”、“OX40多肽”、“OX40表位”等可互换地使用，通常是指源自OX40蛋白(例如人OX40)的多肽片段，其能够被OX40特异性抗体识别，与OX40特异性抗体结合和/或能够产生OX40特异性抗体。在本申请中“OX40抗原多肽”可由OX40蛋白上一段连续的氨基酸残基组成，也可指位于OX40蛋白上不连续位置的氨基酸残基的组合。
在本申请中，术语“免疫原性”通常是指能够刺激机体形成特异抗体或致敏淋巴细胞的能力。例如，可指抗原多肽能够刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质(例如，抗体)和/或致敏淋巴细胞的特性。术语“免疫原性”也可指抗原多肽刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫反应。
在本申请中，术语“非人生物体”通常是指除人类之外的生命形式。例如，“非人生物体”可包括植物、动物、微生物和/或病毒等，或者它们的有生命特征的部分。所述微生物可以是，例如细菌。所述动物可以是，例如脊椎动物(如哺乳动物)。在某些实施方式中，“非人生物体”包括非人灵长类动物、山羊、绵羊、狗、牛、鸡、两栖动物、爬行动物、大鼠、小鼠等。
在本申请中，术语“疫苗”通常是指用各类免疫原(例如，病原体或抗原多肽片段等)制作的、用于预防接种和/或治疗与该免疫原直接或间接相关的疾病或病症的生物制品。
在本申请中，术语“癌症疫苗”通常是指通过利用肿瘤细胞相关抗原(例如，抗原多肽或其组合)，来激活受试者针对癌症的免疫系统的生物制品。
在本申请中，术语“激动性抗体”通常是指能够激活目标分子和/或增强其活性的抗体分子或其抗原结合片段。例如，本申请中的“激动性抗体”可以是特异性结合目标分子的抗体分子或其抗原结合片段，其中，所述抗体分子或其抗原结合片段与目标分子的结合使得目标分子由非活化状态转变为活化状态，或者由低活性状态转变为高活性状态。在本申请中，“OX40激动性抗体”通常指能够特异性识别和/或结合OX40的抗体或其抗原结合片段，且所述抗体或其抗原结合片段特异性识别和/或结合OX40后能够使OX40由非活 化状态转变为活化状态，或者由低活性状态转变为高活性状态。
在本申请中，术语“免疫活性物质”通常是指能够产生免疫应答或者能够使机体产生特异性免疫反应的物质。免疫活性物质可以由效应细胞(例如，T细胞)或者其它细胞产生。常见的免疫活性物质可包括，例如抗体或其抗原结合片段、趋化因子、生长因子、免疫佐剂、细胞因子(例如干扰素、白介素、淋巴因子等)、细胞激素、炎性因子、病毒或其组成部分、细菌或其组成部分等。所述免疫应答或特异性免疫反应可包括，例如，增强抗体产生、增强体液应答、增强细胞免疫应答等。
在本申请中，术语“OX40-OX40L六聚体”通常是指由OX40与其配体OX40L构成的蛋白六聚体。例如，在本申请中，OX40-OX40L六聚体中可包含3个OX40蛋白分子以及3个OX40L蛋白分子。在某些实施方式中，每个OX40蛋白分子与2个OX40L蛋白分子结合，且每个OX40L蛋白分子与2个OX40蛋白分子结合。在某些实施方式中，OX40蛋白分子与OX40L蛋白分子交替排列而形成所述OX40-OX40L六聚体。
在本申请中，术语“激动剂”通常是指能够激活目标分子和/或增强其活性的物质或试剂。例如，本申请中的“激动剂”可以是激动性抗体或其它物质，所述激动剂可通过直接或间接与目标分子相结合而使目标分子由非活化状态转变为活化状态，或者由低活性状态转变为高活性状态。在本申请中，“OX40激动剂”通常指能够激活OX40和/或增强其活性的物质或试剂。例如，OX40激动剂可通过直接或间接与OX40相结合而使OX40由非活化状态转变为活化状态，或者由低活性状态转变为高活性状态。
在本申请中，术语“阻止OX40-OX40L六聚体形成”通常是指使得OX40与其配体OX40L之间无法形成六聚体结构。在某些情形中，OX40与OX40L仍能够彼此结合，但是无法形成六聚体(例如，仅形成2聚体、3聚体、4聚体等)。在某些情形中，OX40与OX40L无法彼此结合或者结合后无法处于有活性的分子构象。
在本申请中，术语“序列同源性”通常是指两个或更多个多核苷酸序列间或者两个或更多个多肽序列间的序列相似性或可互换性。当使用计算机程序或软件(例如，Emboss Needle或BestFit)来确定不同氨基酸序列间的序列同一性、相似性或同源性时，可使用默认的参数设置。也可以选择适当的记分矩阵，例如blosum45或blosum80,来优化同一性、相似性或同源性分数。在某些实施方式中，同源的多核苷酸包括下述多核苷酸：其能够在严紧条件下与对照多核苷酸序列杂交并且与对照多核苷酸序列相比具有至少60％、至少65％、至少70％、至少80％、至少90％、至少95％、至少97％、至少98％、至少99％甚至至少100％序列同一性。同源的多肽可以是下述多肽：当在优化条件下进行序列比对时，其与对照多肽序列具有至少80％、至少85％、至少90％、至少95％、至少97％、至少98％、至少99％、甚至至少100％的序列同一性。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用 BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“融合蛋白”通常是指下述多肽分子，其中一种多肽(例如本申请的OX40抗原多肽)的氨基酸序列直接或间接地(例如通过连接子)与另一种异源多肽的氨基酸序列相融合(例如，通过肽键)。
在本申请中，术语“免疫缀合物”通常是指通过将多肽(例如，本申请的OX40抗原多肽)与抗体或其片段(例如，抗体恒定区或抗体Fc区)相缀合而形成的蛋白类分子。
OX40抗原多肽、融合蛋白或免疫缀合物
一方面，本申请提供了一种分离的OX40抗原多肽。本申请所述的分离的OX40抗原多肽可包含SEQ ID NO:1-32以及它们的片段或变体中的任一项或多项所示的氨基酸序列。所述氨基酸序列的变体可包含：1)与SEQ ID NO:1-32中任一项所示的氨基酸序列具有至少80％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同源性的氨基酸序列；和/或2)在SEQ ID NO:1-32中任一项所示的氨基酸序列中经过取代、缺失或添加一个或几个(例如，1-2个，1-3个，1-4个，1-5个，1-6个，1-7个，1-8个，1-9个，1-10个，1-11个，1-12个或更多)氨基酸而获得的氨基酸序列。
在本申请中，所述氨基酸的取代可以是保守氨基酸取代或非保守氨基酸取代。经取代后的所述OX40抗原多肽仍能够被OX40激动性抗体(例如，DF004或其抗原结合片段)识别或结合(例如，特异性识别或结合)，或者仍能够产生具有OX40激动性抗体(例如，DF004或其抗原结合片段)的一个或多个特征的抗体。
例如，所述氨基酸取代可以为非保守取代。所述非保守取代可包括以非保守的形式改变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有不同侧链大小或不同特性(例如，疏水性)的氨基酸残基。
所述氨基酸取代也可以为保守取代。所述保守取代可包括以保守的形式改变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有相同或相似侧链大小或者相同或相似特性(例如，仍为亲水性)的氨基酸残基。这样的保守取代通常不会对所产生的蛋白质的结构或功能带来很大影响。在本申请中，作为所述分离的OX40抗原多肽的氨基酸序列变体可包括不显著改变蛋白质结构或其功能(例如，产生或结合OX40激动性抗体的能力)的保守氨基酸取代。
作为示例，下述各组中每组内各氨基酸间的相互取代在本申请中可被认为是保守取代：
具有非极性侧链的氨基酸组：丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸和甲硫氨酸。
不带电荷、具有极性侧链的氨基酸组：甘氨酸、丝氨酸、苏氨酸，半胱氨酸，酪氨酸，天冬酰胺和谷氨酰胺。
带负电荷、具有极性侧链的氨基酸组：天冬氨酸和谷氨酸。
带正电荷的碱性氨基酸：赖氨酸、精氨酸和组氨酸。
带苯基的氨基酸：苯丙氨酸、色氨酸和酪氨酸。
在某些实施方式中，所述氨基酸序列的变体不包含突变G70A和/或突变F71A。在某些实施方式中，所述氨基酸序列的变体不包含残基G70和/或残基F71处的氨基酸突变。在本申请中，残基G70是指相应于SEQ ID NO:33所示氨基酸序列中第70位的残基G，残基F71是指相应于SEQ ID NO:33所示氨基酸序列中第71位的残基F。突变G70A是指将相应于SEQ ID NO:33所示氨基酸序列中第70位的残基G突变为A。突变F71A是指将相应于SEQ ID NO:33所示氨基酸序列中第71位的残基F突变为A。例如，所述OX40抗原多肽可源自OX40(例如人OX40)，且其在相应于SEQ ID NO:33所示氨基酸序列中第70位的残基G处不包含氨基酸突变。例如，所述OX40抗原多肽可源自OX40(例如人OX40)，且其在相应于SEQ ID NO:33所示氨基酸序列中第71位的残基F处不包含氨基酸突变。
在本申请中，某氨基酸序列中的某残基“相应于”另一氨基酸序列中的某残基通常是指，在优化条件下进行氨基酸序列比对时所获得的残基对应关系。所述序列比对可通过本领域技术人员了解的方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。例如，SEQ ID NO:2所示氨基酸序列中第1位至第7位的残基分别相应于SEQ ID NO:33所示氨基酸序列中第65位至第71位的残基。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：X 1PCX 2X 3GF(SEQ ID NO:1)，其中X 1可以是R、A或H，X 2可以是G或E，且X 3可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列RPCGPGF(SEQ ID NO:2)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：X 1PCX 2X 3GFYNX 4X 5VX 6X 7X 8X 9C(SEQ ID NO:3)，其中X 1可以是R、A或H，X 2可以是G或E，X 3可以是P或T，X 4可以是D或E，X 5可以是V或A，X 6可以是S或N，X 7可以是S或Y，X 8可以是K或D，X 9可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列RPCGPGFYNDVVSSKPC(SEQ ID NO:4)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：X 1PCX 2X 3GFYNX 4X 5VX 6X 7X 8X 9CKX 10CTX 11CN(SEQ ID NO:5)，其中X 1可以是R、A或H，X 2可以是 G或E，X 3可以是P或T，X 4可以是D或E，X 5可以是V或A，X 6可以是S或N，X 7可以是S或Y，X 8可以是K或D，X 9可以是P或T，X 10可以是P、A或Q，且X 11可以是W、A或Q。例如，所述分离的OX40抗原多肽可包含氨基酸序列RPCGPGFYNDVVSSKPCKPCTWCN(SEQ ID NO:6)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：X 1PCX 2X 3GFYNX 4X 5VX 6X 7X 8X 9CKX 10CTX 11CNX 12RSGSEX 13KQX 14CTX 15TX 16DTVC(SEQ ID NO:7)，其中X 1可以是R、A或H，X 2可以是G或E，X 3可以是P或T，X 4可以是D或E，X 5可以是V或A，X 6可以是S或N，X 7可以是S或Y，X 8可以是K或D，X 9可以是P或T，X 10可以是P、A或Q，X 11可以是W、A或Q，X 12可以是L或H，X 13可以是R或L，X 14可以是L或N，X 15可以是A或P，且X 16可以是Q或E。例如，所述分离的OX40抗原多肽可包含氨基酸序列RPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC(SEQ ID NO:8)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：GMVSRCX 1X 2X 3X 4X 5TX 6CX 7PCX 8X 9GF(SEQ ID NO:9)，其中X 1可以是S或D，X 2可以是R或H，X 3可以是S或T，X 4可以是Q或R，X 5可以是N或D，X 6可以是V或L，X 7可以是R、A或H，X 8可以是G或E，且X 9可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列GMVSRCSRSQNTVCRPCGPGF(SEQ ID NO:10)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：CX 1ECX 2PGX 3GMVSRCX 4X 5X 6X 7X 8TX 9CX 10PCX 11X 12GF(SEQ ID NO:11)，其中X 1可以是H或R，X 2可以是R或Q，X 3可以是N或H，X 4可以是S或D，X 5可以是R或H，X 6可以是S或T，X 7可以是Q或R，X 8可以是N或D，X 9可以是V或L，X 10可以是R、A或H，X 11可以是G或E，且X 12可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列CHECRPGNGMVSRCSRSQNTVCRPCGPGF(SEQ ID NO:12)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：LX 1CVX 2X 3TYPSX 4X 5X 6CCX 7ECX 8PGX 9GMVSRCX 10X 11X 12X 13X 14TX 15CX 16PCX 17X 18GF(SEQ ID NO:13)，其中X 1可以是H或N，X 2可以是G或K，X 3可以是D或H，X 4可以是N或G，X 5可以是D或H，X 6可以是R或K，X 7可以是H或R，X 8可以是R或Q，X 9可以是N或H，X 10可以是S或D，X 11可以是R或H，X 12可以是S或T，X 13可以是Q或R，X 14可以是N或D，X 15可以是V或L，X 16可以是R、A或H，X 17可以是G或E，且X 18可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGF(SEQ ID NO:14)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列： GMVSRCX 1X 2X 3X 4X 5TX 6CX 7PCX 8X 9GFYNX 10X 11VX 12X 13X 14X 15C(SEQ ID NO:15)，其中X 1可以是S或D，X 2可以是R或H，X 3可以是S或T，X 4可以是Q或R，X 5可以是N或D，X 6可以是V或L，X 7可以是R、A或H，X 8可以是G或E，X 9可以是P或T，X 10可以是D或E，X 11可以是V或A，X 12可以是S或N，X 13可以是S或Y，X 14可以是K或D，且X 15可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列GMVSRCSRSQNTVCRPCGPGFYNDVVSSKPC(SEQ ID NO:16)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：CX 1ECX 2PGX 3GMVSRCX 4X 5X 6X 7X 8TX 9CX 10PCX 11X 12GFYNX 13X 14VX 15X 16X 17X 18C(SEQ ID NO:17)，其中X 1可以是H或R，X 2可以是R或Q，X 3可以是N或H，X 4可以是S或D，X 5可以是R或H，X 6可以是S或T，X 7可以是Q或R，X 8可以是N或D，X 9可以是V或L，X 10可以是R、A或H，X 11可以是G或E，X 12可以是P或T，X 13可以是D或E，X 14可以是V或A，X 15可以是S或N，X 16可以是S或Y，X 17可以是K或D，且X 18可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列CHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPC(SEQ ID NO:18)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：LX 1CVX 2X 3TYPSX 4X 5X 6CCX 7ECX 8PGX 9GMVSRCX 10X 11X 12X 13X 14TX 15CX 16PCX 17X 18GFYNX 19X 20VX 21X 2 2X 23X 24C(SEQ ID NO:19)，其中X 1可以是H或N，X 2可以是G或K，X 3可以是D或H，X 4可以是N或G，X 5可以是D或H，X 6可以是R或K，X 7可以是H或R，X 8可以是R或Q，X 9可以是N或H，X 10可以是S或D，X 11可以是R或H，X 12可以是S或T，X 13可以是Q或R，X 14可以是N或D，X 15可以是V或L，X 16可以是R、A或H，X 17可以是G或E，X 18可以是P或T，X 19可以是D或E，X 20可以是V或A，X 21可以是S或N，X 22可以是S或Y，X 23可以是K或D，且X 24可以是P或T。例如，所述分离的OX40抗原多肽可包含氨基酸序列LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPC(SEQ ID NO:20)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：GMVSRCX 1X 2X 3X 4X 5TX 6CX 7PCX 8X 9GFYNX 10X 11VX 12X 13X 14X 15CKX 16CTX 17CN(SEQ ID NO:21)，其中X 1可以是S或D，X 2可以是R或H，X 3可以是S或T，X 4可以是Q或R，X 5可以是N或D，X 6可以是V或L，X 7可以是R、A或H，X 8可以是G或E，X 9可以是P或T，X 10可以是D或E，X 11可以是V或A，X 12可以是S或N，X 13可以是S或Y，X 14可以是K或D，X 15可以是P或T，X 16可以是P、A或Q，且X 17可以是W、A或Q。例如，所述分离的OX40抗原多肽可包含氨基酸序列GMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCN(SEQ ID NO:22)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列： CX 1ECX 2PGX 3GMVSRCX 4X 5X 6X 7X 8TX 9CX 10PCX 11X 12GFYNX 13X 14VX 15X 16X 17X 18CKX 19CTX 20CN(SEQ ID NO:23)，其中X 1可以是H或R，X 2可以是R或Q，X 3可以是N或H，X 4可以是S或D，X 5可以是R或H，X 6可以是S或T，X 7可以是Q或R，X 8可以是N或D，X 9可以是V或L，X 10可以是R、A或H，X 11可以是G或E，X 12可以是P或T，X 13可以是D或E，X 14可以是V或A，X 15可以是S或N，X 16可以是S或Y，X 17可以是K或D，X 18可以是P或T，X 19可以是P、A或Q，且X 20可以是W、A或Q。例如，所述分离的OX40抗原多肽可包含氨基酸序列CHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCN(SEQ ID NO:24)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：LX 1CVX 2X 3TYPSX 4X 5X 6CCX 7ECX 8PGX 9GMVSRCX 10X 11X 12X 13X 14TX 15CX 16PCX 17X 18GFYNX 19X 20VX 21X 2 2X 23X 24CKX 25CTX 26CN(SEQ ID NO:25)，其中X 1可以是H或N，X 2可以是G或K，X 3可以是D或H，X 4可以是N或G，X 5可以是D或H，X 6可以是R或K，X 7可以是H或R，X 8可以是R或Q，X 9可以是N或H，X 10可以是S或D，X 11可以是R或H，X 12可以是S或T，X 13可以是Q或R，X 14可以是N或D，X 15可以是V或L，X 16可以是R、A或H，X 17可以是G或E，X 18可以是P或T，X 19可以是D或E，X 20可以是V或A，X 21可以是S或N，X 22可以是S或Y，X 23可以是K或D，X 24可以是P或T，X 25可以是P、A或Q，且X 26可以是W、A或Q。例如，所述分离的OX40抗原多肽可包含氨基酸序列LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCN(SEQ ID NO:26)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：GMVSRCX 1X 2X 3X 4X 5TX 6CX 7PCX 8X 9GFYNX 10X 11VX 12X 13X 14X 15CKX 16CTX 17CNX 18RSGSEX 19KQX 20CTX 2 1TX 22DTVC(SEQ ID NO:27)，其中X 1可以是S或D，X 2可以是R或H，X 3可以是S或T，X 4可以是Q或R，X 5可以是N或D，X 6可以是V或L，X 7可以是R、A或H，X 8可以是G或E，X 9可以是P或T，X 10可以是D或E，X 11可以是V或A，X 12可以是S或N，X 13可以是S或Y，X 14可以是K或D，X 15可以是P或T，X 16可以是P、A或Q，X 17可以是W、A或Q，X 18可以是L或H，X 19可以是R或L，X 20可以是L或N，X 21可以是A或P，且X 22可以是Q或E。例如，所述分离的OX40抗原多肽可包含氨基酸序列GMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC(SEQ ID NO:28)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：CX 1ECX 2PGX 3GMVSRCX 4X 5X 6X 7X 8TX 9CX 10PCX 11X 12GFYNX 13X 14VX 15X 16X 17X 18CKX 19CTX 20CNX 21RSG SEX 22KQX 23CTX 24TX 25DTVC(SEQ ID NO:29)，其中X 1可以是H或R，X 2可以是R或Q，X 3可以是N 或H，X 4可以是S或D，X 5可以是R或H，X 6可以是S或T，X 7可以是Q或R，X 8可以是N或D，X 9可以是V或L，X 10可以是R、A或H，X 11可以是G或E，X 12可以是P或T，X 13可以是D或E，X 14可以是V或A，X 15可以是S或N，X 16可以是S或Y，X 17可以是K或D，X 18可以是P或T，X 19可以是P、A或Q，X 20可以是W、A或Q，X 21可以是L或H，X 22可以是R或L，X 23可以是L或N，X 24可以是A或P，且X 25可以是Q或E。例如，所述分离的OX40抗原多肽可包含氨基酸序列CHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC(SEQID NO:30)。
在某些实施方式中，本申请的分离的OX40抗原多肽包含氨基酸序列：LX 1CVX 2X 3TYPSX 4X 5X 6CCX 7ECX 8PGX 9GMVSRCX 10X 11X 12X 13X 14TX 15CX 16PCX 17X 18GFYNX 19X 20VX 21X 2 2X 23X 24CKX 25CTX 26CNX 27RSGSEX 28KQX 29CTX 30TX 31DTVC(SEQ ID NO:31)，其中X 1可以是H或N，X 2可以是G或K，X 3可以是D或H，X 4可以是N或G，X 5可以是D或H，X 6可以是R或K，X 7可以是H或R，X 8可以是R或Q，X 9可以是N或H，X 10可以是S或D，X 11可以是R或H，X 12可以是S或T，X 13可以是Q或R，X 14可以是N或D，X 15可以是V或L，X 16可以是R、A或H，X 17可以是G或E，X 18可以是P或T，X 19可以是D或E，X 20可以是V或A，X 21可以是S或N，X 22可以是S或Y，X 23可以是K或D，X 24可以是P或T，X 25可以是P、A或Q，X 26可以是W、A或Q，X 27可以是L或H，X 28可以是R或L，X 29可以是L或N，X 30可以是A或P，且X 31可以是Q或E。例如，所述分离的OX40抗原多肽可包含氨基酸序列LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC(SEQ ID NO:32)。
在某些实施方式中，本申请所述的分离的OX40抗原多肽包含约4-160个(例如，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，至少15个，至少20个，至少25个，至少30个，至少35个，至少40个，至少45个，至少50个，至少55个，至少60个，至少65个，至少70个，至少75个，至少80个，至少85个，至少90个，至少95个，至少100个，至少110个，至少120个，至少130个，至少140个，至少150个，至少160个或更多个)氨基酸残基。
例如，本申请所述的分离的OX40抗原多肽可源自OX40的细胞外结构域，其片段或变体。例如，其可源自人、食蟹猴、小鼠或大鼠OX40的细胞外结构域，其片段或变体。在某些实施方式中，本申请所述的分离的OX40抗原多肽源自人OX40。例如，其可源自人OX40的细胞外结构域，其片段或变体。所述人OX40细胞外结构域的氨基酸序列可如SEQ ID NO:43所示。
例如，所述OX40抗原多肽可包含OX40(如人OX40)的细胞外结构域、其变体或部分，如一个或多 个(例如，1个、2个、3个或4个)半胱氨酸富集域(CRD)、其变体或部分。
在某些实施方式中，本申请的分离的OX40抗原多肽源自OX40(例如，人OX40)细胞外结构域中包含下组中一个或多个结构域或其片段的区域：半胱氨酸富集域1(CRD1)、半胱氨酸富集域2(CRD2)和半胱氨酸富集域3(CRD3)。在某些实施方式中，本申请的分离的OX40抗原多肽源自OX40(例如，人OX40)细胞外结构域中包含下组中一个或多个结构域或其片段的区域：半胱氨酸富集域1(CRD1)和半胱氨酸富集域2(CRD2)。在某些实施方式中，本申请的分离的OX40抗原多肽不包含OX40(例如，人OX40)细胞外结构域中的下列区域：半胱氨酸富集域3(CRD3)或半胱氨酸富集域4(CRD4)。
在某些实施方式中，所述OX40抗原多肽包含OX40(例如人OX40)细胞外结构域中的CRD2区、其变体或其片段。在某些实施方式中，所述OX40抗原多肽还包含OX40(例如人OX40)细胞外结构域中的CRD1区、其变体或其片段。
在某些实施方式中，所述OX40抗原多肽不包含OX40(例如人OX40)细胞外结构域中的CRD3区或其片段。在某些实施方式中，所述OX40抗原多肽不包含OX40(例如人OX40)细胞外结构域中的CRD4区或其片段。
例如，本申请的分离的OX40抗原多肽可包含源自下组中的一个或多个结构域或其片段的区域：1)OX40(例如，人OX40)的CRD1的模块A1；2)OX40(例如，人OX40)的CRD1的模块B2；3)OX40(例如，人OX40)的CRD2的模块A1；和4)OX40(例如，人OX40)的CRD2的模块B2。在某些实施方式中，本申请的分离的OX40抗原多肽包含OX40(例如，人OX40)的CRD1的模块A1、其变体或片段。在某些实施方式中，本申请的分离的OX40抗原多肽包含OX40(例如，人OX40)的CRD1的模块B2、其变体或片段。在某些实施方式中，本申请的分离的OX40抗原多肽包含OX40(例如，人OX40)的CRD2的模块A1、其变体或片段。在某些实施方式中，本申请的分离的OX40抗原多肽包含OX40(例如，人OX40)的CRD2的模块B2、其变体或片段。
例如，人OX40的CRD1中模块A1的氨基酸序列可如SEQ ID NO:45所示，人OX40的CRD1中模块B2的氨基酸序列可如SEQ ID NO:46所示，人OX40的CRD1的氨基酸序列可如SEQ ID NO:47所示。人OX40的CRD2中模块A1的氨基酸序列可如SEQ ID NO:48所示，人OX40的CRD2中模块B2的氨基酸序列可如SEQ ID NO:49所示，人OX40的CRD2的氨基酸序列可如SEQ ID NO:50所示。
此外，人OX40的CRD3的氨基酸序列可如SEQ ID NO:51所示。人OX40的CRD4的氨基酸序列可如SEQ ID NO:52所示。
本申请的分离的OX40抗原多肽可具有免疫原性。例如，所述OX40抗原多肽能够刺激机体形成OX40特异性抗体和/或致敏淋巴细胞。
另一方面，本申请还提供了包含所述分离的OX40抗原多肽的融合蛋白或免疫缀合物。例如，可将所述分离的OX40抗原多肽与一种或多种异源多肽(例如，抗体或抗体的组成部分，或者其它抗原多肽)相融合，而得到所述融合蛋白或免疫缀合物。其中，用于与所述分离的OX40抗原多肽相融合的异源多肽可包含抗体(例如，IgG抗体)的恒定区或Fc区。在某些实施方式中，用于与所述分离的OX40抗原多肽相融合的异源多肽可包含其它抗原多肽，例如源自OX40(如人OX40)的其它抗原多肽。本申请所述的分离的OX40抗原多肽可直接(例如，在框内)或间接(例如，通过连接子，如肽连接子)与所述一种或多种异源多肽相连接、缀合或融合。可通过本领域技术人员已知的方法制备所述融合蛋白或免疫缀合物，例如可通过分子克隆和蛋白质重组表达的方法制备所述融合蛋白或免疫缀合物。
核酸、载体、细胞和制备方法
在另一个方面，本申请提供了一种或多种分离的核酸分子，所述核酸分子可编码本申请所述的分离的OX40抗原多肽和/或本申请所述的融合蛋白或免疫缀合物。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述OX40抗原多肽和/或所述融合蛋白或免疫缀合物的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience,New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，所述载体可包含本申请所述的一种或多种分离的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA 启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。在某些实施方式中，所述载体为表达载体。
在另一个方面，本申请提供了一种或多种细胞(例如，宿主细胞)或非人生物体，其可包含或表达本申请所述的OX40抗原多肽、本申请所述的融合蛋白或免疫缀合物、本申请所述的一种或多种分离的核酸分子、或者本申请所述的一种或多种载体。例如，可将本申请所述的载体引入所述细胞(例如，宿主细胞)中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞或HEK293A细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。所述非人生物体可以为转基因的或遗传工程化的生物体，其可源自所述细胞(例如，宿主细胞)或者包含所述细胞(例如，宿主细胞)。
在另一个方面，本申请提供了制备所述OX40抗原多肽或者所述融合蛋白或免疫缀合物的方法。所述方法可包括，在使得所述OX40抗原多肽或者所述融合蛋白或免疫缀合物表达的条件下，培养本申请所述的细胞(例如，宿主细胞)。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括分离和/或纯化所述OX40抗原多肽或者所述融合蛋白或免疫缀合物的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的OX40抗原多肽或者所述的融合蛋白或免疫缀合物。
组合物和疫苗
在另一个方面，本申请提供了一种组合物(例如药物组合物)，其可包含本申请所述的OX40抗原多肽、本申请所述的融合蛋白或免疫缀合物、本申请所述的核酸分子、本申请所述的载体、或本申请所述的细胞(例如，宿主细胞)，以及任选地药学上可接受的佐剂。
所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，可按照本领域的常规技术手段将所述药物组合物与可药用载体或稀释剂以及任何其他已知的辅剂和赋形剂配制在一起，例如按照Remington：The Science and Practice of Pharmacy，第十九版，Gennaro编辑，Mack Publishing Co.，Easton，PA，1995中公开的技术进行操作。
所述药物组合物可以用于在体内(例如，生物体内，如动物体内或人体内)或体外(例如分离的细胞 或组织中)产生免疫活性物质。所述免疫活性物质可包括OX40特异性抗体，例如OX40激动性抗体，例如抗体DF004。在某些实施方式中，所述药物组合物用于抑制肿瘤生长(例如，血液瘤或实体肿瘤)。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
例如，本申请所述的药物组合物可以为适于口服给药的形式，如片剂，胶囊剂，丸剂，粉剂，缓释制剂，溶液剂，混悬剂，或者用于肠胃外注射，如无菌溶液剂，混悬剂或乳剂，或者用于软膏或乳膏局部给药或作为栓剂直肠给药。所述药物组合物可以是适合精确剂量单次给药的单位剂量形式。所述药物组合物可以进一步包含常规的药物载体或赋形剂。此外，所述药物组合物可以包括其他药物或药剂，载体，佐剂等。
本申请所述的药物组合物可以包含治疗有效量的所述OX40抗原多肽或者所述融合蛋白或免疫缀合物。所述治疗有效量是能够产生免疫活性物质(例如，OX40特异性抗体，如OX40激动性抗体)，和/或能够治疗或减缓疾病(例如肿瘤)发展或进展的量。所述剂量的具体量/浓度可以根据施用方法和患者需要而变化，并且可以基于例如患者体积，粘度和/或体重等来确定。应当理解的是，基于特定患者，制剂和/或疾病的状况，本领域技术人员(例如，医生或药剂师)可以方便地调整这些特定剂量。
本申请所述的药物组合物可以为疫苗(例如，癌症疫苗)。因此，在另一个方面，本申请提供了一种疫苗，其可包含一种或多种选自下列的物质以及任选地免疫原性载体：1)一种或多种本申请所述的OX40抗原多肽；2)本申请所述的融合蛋白或免疫缀合物；3)本申请所述的核酸分子；4)本申请所述的载体；5)本申请所述的细胞(例如，宿主细胞)；和6)本申请所述的组合物(例如，药物组合物)。在某些实施方式中，所述疫苗为癌症疫苗。
本申请所述的疫苗(例如，癌症疫苗)还可以包含一种或多种其它的抗原(例如，肿瘤抗原)。所述其它的抗原可以包括一种或多种其它的OX40抗原多肽。所述疫苗可用于治疗受试者(例如，哺乳动物，比如人)中的一种或多种癌症(例如，血液癌或实体瘤，包括与T细胞、特别是CD4 +和/或CD8 +T细胞相关的癌症)。所述癌症可以包括但不限于，例如，黑色素瘤、脑癌、骨癌、白血病、淋巴瘤、上皮细胞癌、腺癌、胃肠道癌(如唇癌、口腔癌、食道癌、小肠癌、胃癌、结肠癌)、肝癌、膀胱癌、胰腺癌、卵巢癌、宫颈癌、肺癌、乳腺癌、皮肤癌、鳞状细胞癌、前列腺癌和/或肾细胞癌等。
可以通过化学合成或重组表达、纯化等方法制备本申请所述的分离的OX40抗原多肽或者所述融合蛋白或免疫缀合物，随后可将所述OX40抗原多肽或者所述融合蛋白或免疫缀合物与一种或多种其它肿瘤抗原和/或药学上可接受的佐剂进行混合而制备所述疫苗(例如，癌症疫苗)。
在某些情形中，所述疫苗中可包含一种或多种免疫原性载体或佐剂。例如，所述OX40抗原多肽、所 述融合蛋白或免疫缀合物或其它肿瘤抗原可与一种或多种糖脂类似物相组合或结合，以增强其免疫效果。
这些疫苗或组合物可被用于免疫(例如，通过肌内或肠胃外施用)受试者(例如，动物或人，如鼠类、兔类、羊类等)。所述疫苗中也可包含一种或多种药学上可接受的佐剂，例如缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
抗体及其产生、筛选方法及用途
在另一个方面，本申请提供了一种抗体，其能够特异性结合本申请所述的OX40抗原多肽。例如，所述抗体可以为OX40激动性抗体。在某些实施方式中，所述抗体为单克隆抗体。在某些实施方式中，所述抗体基本上不结合包含突变G70A和/或突变F71A的OX40(例如，人OA40)变体。例如，所述抗体可基本上不结合氨基酸序列如SEQ ID NO:35或SEQ ID NO:36所示的人OX40变体。
在某些实施方式中，所述抗体与DF004竞争结合本申请的所述OX40抗原多肽。在某些情形中，所述抗体可以不是DF004。
本申请所述的抗体DF004包含轻链和重链，所述轻链包含LCDR1、LCDR2和LCDR3，其中所述LCDR1的氨基酸序列如SEQ ID NO:63所示，所述LCDR2的氨基酸序列如SEQ ID NO:64所示且所述LCDR3的氨基酸序列如SEQ ID NO:65所示。所述重链包含HCDR1、HCDR2和HCDR3，其中所述HCDR1的氨基酸序列如SEQ ID NO:66所示，所述HCDR2的氨基酸序列如SEQ ID NO:67所示且所述HCDR3的氨基酸序列如SEQ ID NO:68所示。
本申请所述的抗体DF004的重链可变区氨基酸序列如SEQ ID NO:70所示，且其轻链可变区氨基酸序列如SEQ ID NO:69所示。
本申请所述的抗体DF004的重链氨基酸序列如SEQ ID NO:56所示，且其轻链氨基酸序列如SEQ ID NO:58所示。
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗体(例如，OX40激动性抗体)，以及任选地药学上可接受的佐剂。所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在另一个方面，本申请提供了一种用于产生OX40激动性抗体的方法。所述方法可以包括施用(例如，向受试者，如待免疫的动物或人；或者在体外，例如向分离的细胞或组织)本申请所述的OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物。例如，所述方法可以包括用本申请所述的OX40抗原多肽免疫动物。在某些实施方式中，所述方法还包括分离与所述OX40抗原多肽特异性结合的抗体。
在另一个方面，本申请提供了本申请所述OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物用于制备免疫活性物质的用途，所述免疫活性物质可包括OX40激动性抗体。所产生的OX40激动性抗体可用于治疗疾病(例如，癌症，如血液癌或实体瘤，特别是与OX40的表达或调控相关的癌症)。因此，在某些实施方式中，本申请提供了所述OX40抗原多肽或者所述融合蛋白或免疫缀合物用于制备药物的用途，所述药物可用于治疗疾病(例如，癌症，如血液癌或实体瘤，特别是与OX40的表达或调控相关的癌症)。所述癌症可以包括但不限于，例如，黑色素瘤、脑癌、骨癌、白血病、淋巴瘤、上皮细胞癌、腺癌、胃肠道癌(如唇癌、口腔癌、食道癌、小肠癌、胃癌、结肠癌)、肝癌、膀胱癌、胰腺癌、卵巢癌、宫颈癌、肺癌、乳腺癌、皮肤癌、鳞状细胞癌、前列腺癌和/或肾细胞癌等。
能够利用本申请所述的OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物产生、制备、筛选和/或检测的抗体可包括天然产生的抗体以及通过重组方法制备的抗体。这些抗体可包括利用本领域已知的技术制备的多克隆抗体和单克隆抗体。这些抗体可以为OX40激动性抗体，例如，所述抗体能够激活OX40或增强其活性。所述抗体能够被用于治疗和/或预防与OX40和/或OX40L的表达或活性相关的疾病，例如肿瘤(如血液癌或实体瘤)。所述抗体也可被用于免疫测定中，例如用于对肿瘤组织和/或肿瘤微环境中的其它组织或细胞(如免疫细胞，例如表达OX40的免疫细胞)进行表征、识别或标记。
在利用本申请所述的OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物产生或制备抗体时，可采用本领域中常用的抗体制备方法。
例如，当所述产生或制备的抗体为多克隆抗体时，可采用本领域技术人员已知的方法，如Harlow等人，Antibodies:a Laboratory Manual，Cold spring Harbor Laboratory Press,第二版(1988)，其全文在此通过援引而并入。例如，可将本申请所述的OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物注射或以其它方式施用到宿主动物(包括但不限于，兔、小鼠、大鼠等)中，以诱导含有特异性针对所述OX40抗原、融合蛋白或免疫缀合物的多克隆抗体的血清的生成。所述OX40抗原、融合蛋白或免疫缀合物的施用可通过一次或多次注射来进行，需要的时候还可以施用免疫佐剂。依据宿主种类不同，各种佐剂可用于提高免疫应答，其包括但不限于，弗氏试剂(完全或不完全)、无机佐剂(如氢氧化铝)、表面活性物质(如溶血卵磷脂)、复合多元醇、聚阴离子、肽、油乳剂、匙孔血蓝蛋白、二硝基酚以及潜在有用的人佐剂如BCG(卡介苗)和短小棒状杆菌等。其它的可供使用的佐剂的例子包括MPL-TDM佐剂(单磷酸类脂A，合成的海藻糖二霉菌酸脂)。进行免疫的具体方案为本领域技术人员所知，并可通过能诱发所选动物宿主的免疫应答的任意方式进行。
例如，可通过多次皮下或腹膜内注射、或肌肉内或通过静脉输注将免疫原(具有或不具有佐剂)注射至宿主动物中。该免疫原可包括本申请的分离的OX40抗原多肽、本申请的融合蛋白或免疫缀合物等。可 根据多肽的性质(即，疏水性比例、亲水性比例、稳定性、净电荷、等电点等)，将所述免疫原(例如本申请的分离的OX40抗原多肽)与已知的、在待免疫的宿主动物中具有免疫原性的蛋白相缀合。可通过化学偶联、基于融合蛋白的方法、或其它本领域技术人员已知方法来实现所述缀合。所述在待免疫的宿主动物中具有免疫原性的蛋白可包括，例如匙孔血蓝蛋白、卵白蛋白、血清白蛋白、牛甲状腺蛋白、大豆胰蛋白抑制剂和混杂T辅助肽等。
当所述产生或制备的抗体为单克隆抗体时，可采用本领域技术人员已知的方法，例如杂交瘤技术(可参见美国专利4,376,110；Harlow等人，Antibodies:A Laboratory Manual，Cold Spring Harbor Laboratory Press，第二版(1988)以及Hammerling等人，Monoclonal Antibodies and T-Cell Hybridomas，Elsevier(1981))、重组DNA方法或本领域技术人员已知的其它方法进行制备。可用于制备单克隆抗体的方法的其它实例包括但不限于，人B-细胞杂交瘤技术、EBV-杂交瘤技术等。所产生的单克隆抗体可以是任意免疫球蛋白类别，包括IgG、IgM、IgE、IgA、IgD及其任意亚型。可在体外或体内培养用于产生单克隆抗体的杂交瘤。
在杂交瘤模型中，宿主(例如小鼠、人源化小鼠、具有人免疫系统的小鼠、仓鼠、兔、骆驼或任意其它合适的宿主动物)被免疫以诱发淋巴细胞，该淋巴细胞生成或能够生成特异性结合用于免疫的蛋白(例如本申请的分离的OX40抗原多肽，或者本申请的融合蛋白或免疫缀合物)的抗体。备选地，也可在体外对淋巴细胞进行免疫，然后可通过使用合适的融合剂(例如聚乙二醇)将淋巴细胞与骨髓瘤细胞进行融合，以形成杂交瘤。
一般而言，在制备用于产生抗体的杂交瘤时，如需要人源细胞，可使用外周血淋巴细胞(“PBL”)。如需要非人哺乳动物源细胞，可使用脾细胞或淋巴结细胞。然后，可通过使用合适的融合剂(例如聚乙二醇)将淋巴细胞与永生化细胞系相融合，以形成杂交瘤。永生化细胞系通常为被转化的哺乳动物细胞，特别是啮齿类动物、牛或人源骨髓瘤细胞。例如，可采用大鼠或小鼠的骨髓瘤细胞系。可在适当的培养基中培养所述杂交瘤细胞。例如，所述培养基可包含一种或多种抑制未融合的永生化细胞生长或存活的物质。例如，如果该亲代细胞缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，用于该杂交瘤的培养基通常会包含次黄嘌呤、氨基蝶呤和胸苷(“HAT培养基”)，它们是阻止HGPRT-缺陷型细胞生长的物质。
例如，所使用的永生化细胞系可以是能够有效融合、支持所选抗体生成细胞稳定、高水平地产生抗体、并对培养基(例如HAT培养基)敏感的细胞系。这些骨髓瘤细胞系可包括鼠骨髓瘤细胞系，例如MOPC-21和MPC-11小鼠肿瘤细胞系，源自SP2/0的细胞系等。还可使用人骨髓瘤、鼠-人异源骨髓瘤细胞系、小鼠杂交瘤细胞系NSO等来制备抗体。
可通过免疫沉淀、放射性免疫测定(RIA)、酶联免疫吸附测定(ELISA)等体外结合试验来检测所产 生的单克隆抗体与所述分离的OX40抗原多肽、所述融合蛋白或免疫缀合物的结合亲和力和/或结合特异性。这些技术在本领域技术人员的技能范围内。
当鉴定出能够生成具有所需结合特异性、亲和性和/或活性的抗体的杂交瘤后，可将该克隆通过有限稀释法进行亚克隆并以标准方法培养。合适的培养基可包括，例如，DMEM或RPMI-1640培养基。然后，可使用常规的免疫球蛋白纯化技术(例如，蛋白A-琼脂糖、羟基磷灰石层析、凝胶排阻层析、凝胶电泳、透析或亲和层析)从细胞培养物、宿主动物腹水或血清中分离由该亚克隆分泌的单克隆抗体。
也可通过其它方法，例如重组DNA方法制备或产生所述单克隆抗体。例如，可将由杂交瘤分离的DNA插入表达载体中，随后将其转染到宿主细胞(例如大肠杆菌细胞、NSO细胞、猿COS细胞、中国仓鼠卵巢(CHO)细胞或者其它情况下不会生成免疫球蛋白的骨髓瘤细胞)中，从而在该重组宿主细胞中合成所述单克隆抗体。
在另一个方面，本申请提供了一种阻止OX40-OX40L六聚体形成的方法，所述方法包括施用与本申请所述的OX40抗原多肽特异性结合的试剂。例如，在某些情形中，在体内或体外施用与本申请所述的OX40抗原多肽特异性结合的试剂后，OX40与OX40L无法再结合。在某些情形中，在体内或体外施用与本申请所述的OX40抗原多肽特异性结合的试剂后，OX40与OX40L仍能够彼此结合，但是无法形成六聚体(例如，仅行成2聚体、3聚体、4聚体等)。在某些情形中，OX40与OX40L无法彼此结合或者结合后无法处于有活性的分子构象。例如，与本申请所述的OX40抗原多肽特异性结合的试剂可以为OX40的激动剂。在某些实施方式中，与本申请所述的OX40抗原多肽特异性结合的试剂为OX40激动性抗体(例如，本申请所述的抗体DF004)。
在另一个方面，本申请提供了所述OX40抗原多肽或者所述融合蛋白或免疫缀合物用于制备试剂的用途，所述试剂可用于阻止OX40-OX40L六聚体的形成。在某些实施方式中，所述试剂能够作为激动剂激活OX40。例如，所述试剂可以包括或可以为OX40激动性抗体(例如，本申请所述的抗体DF004)。
在另一个方面，本申请提供了用于筛选和/或检测OX40激动性抗体的方法。所述方法可包括使待筛选和/或待检测的候选抗体或其抗原结合片段、或者表达所述候选抗体或其抗原结合片段的细胞或细胞分泌物与本申请所述的OX40抗原多肽或本申请所述的融合蛋白或免疫缀合物接触。所述方法还可以包括判断所述候选抗体或其抗原结合片段是否与所述OX40抗原多肽特异性结合，且当检测到所述特异性结合时，将该候选抗体鉴别为OX40激动性抗体。例如，所述OX40激动性抗体可包括DF004。
在另一个方面，本申请提供了所述OX40抗原多肽或者本申请所述的融合蛋白或免疫缀合物用于制备试剂的用途，所述试剂可用于检测、识别和/或筛选OX40激动性抗体。在某些实施方式中，所述OX40激动性抗体可包括DF004。
例如，可通过使待检测、识别和/或筛选的细胞、细胞分泌物、细胞培养混合物，或者包含待筛选/检测的抗体或其抗原结合片段的物质(例如，试剂或混合物)与本申请所述的分离的OX40抗原多肽接触，或者与本申请所述的融合蛋白或免疫缀合物接触，然后，根据其是否能够特异性结合本申请所述的分离的OX40抗原多肽(或者本申请所述融合蛋白或免疫缀合物中的所述OX40抗原多肽)，来判断所述细胞、细胞分泌物、细胞培养混合物或者所述试剂或混合物中是否含有OX40激动性抗体或其抗原结合片段。在某些实施方式中，可通过此方法判断所述细胞、细胞分泌物、细胞培养混合物或者所述试剂或混合物中是否含有抗体DF004或其抗原结合片段。
可通过例如，免疫沉淀、放射性免疫测定(RIA)、酶联免疫吸附测定(ELISA)等体外结合试验来进行所述筛选和/或检测。
在另一个方面，本申请提供了预防和/或治疗受试者中的癌症的方法。所述方法可包括向所述受试者施用有效量的、选自下组的一种或多种物质：1)本申请所述的OX40抗原多肽；2)本申请所述的融合蛋白或免疫缀合物；3)本申请所述的核酸分子；4)本申请所述的载体；5)本申请所述的细胞(例如宿主细胞)；6)本申请所述的药物组合物；和7)本申请所述的疫苗。所述癌症可以是例如血液癌或实体瘤，特别是与OX40的表达或调控相关的癌症。例如，所述癌症可以包括但不限于，黑色素瘤、脑癌、骨癌、白血病、淋巴瘤、上皮细胞癌、腺癌、胃肠道癌(如唇癌、口腔癌、食道癌、小肠癌、胃癌、结肠癌)、肝癌、膀胱癌、胰腺癌、卵巢癌、宫颈癌、肺癌、乳腺癌、皮肤癌、鳞状细胞癌、前列腺癌和/或肾细胞癌等。
下面将结合实施例对本申请所述发明的实施方式进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于示例性地说明本申请所述发明，而不应视为限定本申请所述发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：DF004抗体的制备
构建表达载体
根据专利WO2017063162A1得到抗OX40激活性抗体DF004的重链和轻链的全长氨基酸序列，再利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列，并通过人工合成方法获得DF004的轻链基因，命名为DF004-LC(核苷酸序列如SEQ ID NO:57所示)。按照同样的方法获得DF004的重链基因，命名为DF004-HC(核苷酸序列如SEQ ID NO:55所示)。将合成好的DF004-HC和DF004-LC分别经Thermo公司的HindIII和PmeI双酶切克隆至商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-DF004HC和pcDNA4-DF004LC。
宿主细胞转染和目标蛋白表达
转染前两天，准备600mL经悬浮驯化的HEK293(ATCC，CRL-1573 TM)细胞用于瞬时转染，接种密度为0.8×10 6cells/mL。两天后取细胞悬液，离心后重悬于600mL Freestyle293培养基中。
将上述所得的2个重组质粒pcDNA4-DF004HC和pcDNA4-DF004LC用6mL Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液。将每组质粒/PEI混合物分别加入600mL细胞悬液中，放置在37℃，10％CO 2，90rpm中培养；同时补加50μg/L IGF-1。四小时后再补加600mL EX293培养基，2mM谷氨酰胺和50μg/L IGF-1，135rpm培养。24小时后加3.8mM VPA。
培养5～6天后，收集1200mL的DF004抗体瞬时表达培养上清液，通过ProteinA亲和层析法，初步纯化得到DF004抗体样品。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰的看到目的条带。
实施例2：确定与DF004结合的OX40抗原表位
构建表达载体
从uniport数据库得到人OX40细胞外结构域的氨基酸序列(P43489，第1位到第170位，其氨基酸序列如SEQ ID NO:43所示)，再利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列，并通过人工合成方法获得编码OX40细胞外结构域的核酸分子，命名为OX40。将合成好的OX40核酸分子经Thermo公司的HindIII和AgeI双酶切克隆至商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-OX40-His(其中OX40-His的核苷酸序列如SEQ ID NO:60所示)。以实施例1构建的pcDNA-DF004HC为模板，利用PCR的方法(引物如下：SCDKTH-R：GCCGACCGGTGTGTGTCTTATCGCAGCT；DF004-VH-F：GCCGAAGCTTGCCACCATGGAGACAGACACACTCC；其核苷酸序列分别如SEQ ID NO:71、SEQ ID NO:72所示)获得DF004VH-CH1的序列，该序列经HindIII和AgeI双酶切克隆至商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-DF004VH-CH1-His(其中DF004VH-CH1-His的核苷酸序列如SEQ ID NO:62所示)。
宿主细胞转染和目标蛋白表达
转染前两天，准备600mL经悬浮驯化的HEK293(ATCC，CRL-1573 TM)细胞用于瞬时转染，接种密度为0.8×10 6cells/mL。两天后取细胞悬液，离心后分别重悬于600mL Freestyle293培养基中。
将上述实施例中所得的重组表达载体分为两组，组1为200μg pcDNA4-OX40-His；组2为100ug pcDNA4-DF004-VH-CH1-His+100μg pcDNA4-DF004LC。每组质粒用6mL Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液。将每组质粒/PEI混合物分别加入600mL细胞悬液中，放置在37℃， 10％CO 2，90rpm中培养；同时补加50μg/L IGF-1。四小时后再补加600mL EX293培养基，2mM Glutamine和50μg/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。培养5～6天后，分别收集OX40-His(氨基酸序列如SEQ ID NO:59所示)和DF004Fab(DF004VH-CH1-His+DF004LC，其中DF004VH-CH1-His的氨基酸序列如SEQ ID NO:61所示)表达培养上清液，通过Ni-NTA柱亲和层析法，初步纯化得到OX40蛋白和DF004Fab蛋白。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰的看到目的条带。
OX40-DF004复合物的纯化和结晶
将OX40纯化后蛋白与DF004纯化后蛋白按摩尔比2:1比例混合，浓缩至2ml，进入HiLoad 16/600 Superdex 75prep grade，用缓冲液E(10mM Tris-HCl pH7.4，150mM NaCl)洗脱样品，得到OX40和DF004Fab复合物。将OX40-DF004复合物浓缩至30mg/ml，以蛋白：池液＝200nl：200nl比例筛选LMB板，结晶板置于25℃。晶体于(0.1M酒石酸钠钾，20％PEG 3350)长出晶体。对晶体条件优化，方案如下：0.1M酒石酸钠钾，10％～30％PEG 3350。优化后结果最佳为0.1M酒石酸钠钾，14％PEG 3350。进一步以上述条件为基础，加入甘油优化，方案如下：0.1M酒石酸钠钾，14％PEG 3350，5％～25％甘油。选取最佳条件0.1M酒石酸钠钾，14％PEG 3350，10％甘油，对复合物进行结晶。得到的晶体进行X射线衍射，对得到的衍射数据用CCP4软件进行处理，然后用PyMol软件做出示意图，结果如图2所示。
实施例3：对OX40抗原表位上关键结合位点的鉴定及验证
3.1构建OX40抗原关键结合位点表达载体
以实施例2中构建好的pcDNA4-OX40-His质粒为模板，利用重叠PCR的方法针对关键位点进行点突变，其中的关键位点突变策略分别为：W86A、R65A、P83A、A126V、G70A和F71A。点突变所用的引物见下表。
Figure PCTCN2018080280-appb-000001
Figure PCTCN2018080280-appb-000002
将重叠后的PCR产物分别经Thermo公司的HindIII和AgeI双酶切克隆至商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)中，获得重组质粒DNA即：pcDNA4-OX40W86A，pcDNA4-OX40R65A，pcDNA4-OX40P83A，pcDNA4-OX40A126V，pcDNA4-OX40G70A和pcDNA4-OX40F71A。通过测序验证所构建的上述重组质粒的准确性。
3.2宿主细胞转染和目标蛋白表达
转染前两天，准备6×600mL经悬浮驯化的HEK293(ATCC，CRL-1573 TM)细胞用于瞬时转染，接种密度为0.8×10 6cells/mL。两天后取三份细胞悬液，离心后分别重悬于600mL Freestyle293培养基中。
将实施例3.1中所得的6个重组表达载体用6mL Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液。将每组质粒/PEI混合物分别加入600mL细胞悬液中，放置在37℃，10％CO 2，90rpm中培养；同时补加50μg/L IGF-1。四小时后再补加600mL EX293培养基，2mM Glutamine和50ug/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。
培养5～6天后，收集6×1200mL的OX40抗原瞬时表达培养上清液，通过Ni-NTA亲和层析法，纯化 得到OX40点突变后的抗原，可以清晰的看到目的条带。
这些抗原分别命名为OX40-His W86A、OX40-His R65A、OX40-His P83A、OX40-His G70A、OX40-His F71A和OX40-His A126V，其依次分别包含选自以下组的氨基酸序列：SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：85、SEQ ID NO：86和SEQ ID NO：90。
3.3OX40抗原表位关键氨基酸的验证
以包被缓冲液(50mM Na 2CO 3，NaHCO 3pH9.6)稀释上述OX40-His的6种突变抗原(OX40-His W86A、OX40-His R65A、OX40-His P83A、OX40-His G70A、OX40-His F71A和OX40-His A126V)和野生型抗原OX40-His，稀释后的浓度为5μg/ml，100μl/孔，4℃过夜；洗板后，3％BSA-PBS37℃封闭2小时。洗板后将DF004抗体从10μg/ml开始，3倍稀释，共11个浓度，稀释液(1％BSA-PBS)作对照，37℃孵育2小时；洗板后加入羊抗人IgG-HRP(Goat anti-human IgG-HRP conjugated，Sigma:A0170)，37℃孵育1小时。洗板后加入加可溶性单组分TMB底物显色液，室温避光显色5-10min。加入2N H 2SO 4 50μL/孔，终止显色反应。置MD SpectraMax Plus384酶标仪上读OD 450nm-650nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图4所示。从结果中可以看出从结果中可以看出F71A的突变直接导致了OX40不与DF004结合，G70A突变导致OX40结合DF004的亲和力大幅度降低，其余突变基本上不破坏OX40与DF004的结合。
3.4通过BLI技术评价DF004结合不同OX40变体的动力学特征
以PBST(含有0.05％吐温20的PBS)将DF004抗体稀释至10ug/ml，将各OX40-His抗原变体稀释至500nM、250nM、125nM、62.5nM和31.25nM。分别吸取200μl PBST、DF004抗体稀释液、不同浓度的抗原稀释液和10mM pH1.7Gly-HCl加入96孔板(Greinerbio-one，cat#655209)中，放入Octet K2卡槽中。使用AHC(fortebio，cat#18-5060)传感器，设置程序：运行1、PBST孔100s；2、DF004稀释液孔60s；3、PBST孔100s；4、抗原稀释液80s；5、PBST 300s；6、Gly-HCl 5s；7、Gly-HCl 5s；8、Gly-HCl 5s；9、Gly-HCl 5s；10、Gly-HCl 5s；11、Gly-HCl 5s。用Data Analysis 9.0软件进行数据分析和动力学曲线拟合，结果如图5和图6A-6G所示。图6A-6G依次显示了变体OX40-His A126V、OX40-His P83A、OX40-His R65A、OX40-His W86A、OX40-His F71A、OX40-His G70A和对照组野生型OX40-His的曲线拟合结果。从结果中可以看出变体OX40-His G70A与DF004亲和力大幅下降，变体OX40-His F71A的突变导致不与DF004结合，其余变体不破坏与DF004的结合。
尽管本申请所述发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对某些细节进行各种修改和替换，这些改变均在本申请所述发明的保护范围之内。本申请所述发明的全部范围由所附权利要求及其任何等同实施方案给出。

Claims (18)

  1. 分离的OX40抗原多肽，其包含SEQ ID NO:1-32以及它们的片段或变体中的任一项或多项所示的氨基酸序列，所述变体包含：
    1)与SEQ ID NO:1-32中任一项所示的氨基酸序列具有至少80％序列同源性的氨基酸序列；和/或
    2)在SEQ ID NO:1-32中任一项所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸而获得的氨基酸序列。
  2. 根据权利要求1所述的分离的OX40抗原多肽，其中所述变体不包含突变G70A和/或突变F71A。
  3. 根据权利要求1-2中任一项所述的分离的OX40抗原多肽，其中所述变体不包含残基G70和/或残基F71处的氨基酸突变。
  4. 根据权利要求1-3中任一项所述的分离的OX40抗原多肽，其包含约4-160个氨基酸残基。
  5. 根据权利要求1-4中任一项所述的分离的OX40抗原多肽，其源自OX40的细胞外结构域。
  6. 根据权利要求1-5中任一项所述的分离的OX40抗原多肽，其中所述OX40为人OX40。
  7. 融合蛋白或免疫缀合物，其包含权利要求1-6中任一项所述的分离的OX40抗原多肽。
  8. 分离的核酸分子，其编码权利要求1-6中任一项所述的分离的OX40抗原多肽和/或权利要求7所述的融合蛋白或免疫缀合物。
  9. 载体，其包含权利要求8所述的分离的核酸分子。
  10. 细胞或非人生物体，其包含或表达权利要求1-6中任一项所述的OX40抗原多肽，权利要求7所述的融合蛋白或免疫缀合物，权利要求8所述的核酸分子或权利要求9所述的载体。
  11. 药物组合物，其包含权利要求1-6中任一项所述的OX40抗原多肽，权利要求7所述的融合蛋白或免疫缀合物，权利要求8所述的核酸分子，权利要求9所述的载体，或权利要求10所述的细胞，以及任选地药学上可接受的佐剂。
  12. 疫苗，其包含一种或多种选自下列的物质以及任选地免疫原性载体：
    1)一种或多种权利要求1-6中任一项所述的OX40抗原多肽；
    2)权利要求7所述的融合蛋白或免疫缀合物；
    3)权利要求8所述的核酸分子；
    4)权利要求9所述的载体；
    5)权利要求10所述的细胞；和
    6)权利要求11所述的药物组合物。
  13. 根据权利要求12所述的疫苗，其为癌症疫苗。
  14. 用于产生OX40激动性抗体的方法，其包括用权利要求1-6中任一项所述的OX40抗原多肽免疫动物。
  15. 筛选、识别和/或检测OX40激动性抗体的方法，其包括：
    使待筛选、识别和/或检测的候选抗体或其抗原结合片段、表达所述候选抗体或其抗原结合片段的细胞、或者包含所述候选抗体或其抗原结合片段的混合物与权利要求1-6中任一项所述的OX40抗原多肽或权利要求7所述的融合蛋白或免疫缀合物接触，并且
    判断所述候选抗体或其抗原结合片段是否与所述OX40抗原多肽特异性结合，且当检测到所述特异性结合时，将该候选抗体鉴别为OX40激动性抗体。
  16. 权利要求1-6中任一项所述的OX40抗原多肽或权利要求7所述的融合蛋白或免疫缀合物用于制备药物的用途，其中所述药物用于预防和/或治疗癌症。
  17. 阻止OX40-OX40L六聚体形成的方法，包括施用与权利要求1-6中任一项所述的OX40抗原多肽特异性结合的试剂。
  18. 权利要求1-6中任一项所述的OX40抗原多肽或者权利要求7所述的融合蛋白或免疫缀合物用于制备试剂的用途，所述试剂用于检测、识别和/或筛选OX40激动性抗体。
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