WO2019128708A1 - 分离的抗体或其抗原结合片段及其在肿瘤治疗中的应用 - Google Patents

分离的抗体或其抗原结合片段及其在肿瘤治疗中的应用 Download PDF

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WO2019128708A1
WO2019128708A1 PCT/CN2018/120520 CN2018120520W WO2019128708A1 WO 2019128708 A1 WO2019128708 A1 WO 2019128708A1 CN 2018120520 W CN2018120520 W CN 2018120520W WO 2019128708 A1 WO2019128708 A1 WO 2019128708A1
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antibody
antigen
binding fragment
complementarity determining
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French (fr)
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莫世甫
殷迅
赵静
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南京优迈生物科技有限公司
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Priority to AU2018393315A priority Critical patent/AU2018393315A1/en
Priority to KR1020207021966A priority patent/KR20200106052A/ko
Priority to JP2020555277A priority patent/JP7317858B2/ja
Priority to US16/958,557 priority patent/US11440965B2/en
Priority to EP18893920.1A priority patent/EP3733703A4/en
Publication of WO2019128708A1 publication Critical patent/WO2019128708A1/zh

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    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Definitions

  • the present invention relates to an antibody, and more particularly to an isolated antibody or antigen-binding fragment thereof and its use in the treatment of tumors.
  • OX40 Human OX40 is a 277 aa protein, but its apparent molecular weight is approximately 50 kD due to its glycosylation at positions N146 and N160 [1, 2].
  • OX40 is a type I transmembrane protein whose extracellular domain binds to its natural ligand OX40L (CD252) and the intracellular domain is coupled to multiple signaling pathways for T cell activation.
  • Human OX40 is mainly expressed on activated T cells, including CD4, CD8, Th and Treg cells (reviewed in [3]).
  • T cells including CD4, CD8, Th and Treg cells
  • APC cells can detect the expression of OX40L 1-3 days after antigen stimulation.
  • muscle cells also express OX40L under the stimulation of inflammatory factors [4,5], suggesting that the OX40L-OX40 signaling pathway may act extensively on the body's inflammatory response.
  • OX40/OX40L mainly expressed in antigen-activated T cells
  • Activation of antigen-dependent OX40L/OX40 costimulatory molecules is coupled to multiple signaling pathways within T cells.
  • Crystal structure studies indicate that the binding of OX40L to OX40 induces the trimerization of the OX40-OX40L complex [6], thereby forming a binding site for receptor-associated factor (TRAF) in the cell.
  • the latter (TRAF2, 5) can further activate the NF- ⁇ B signaling pathway and inhibit T cell apoptosis [5,7,8].
  • TCR and OX40 on T cells can also synergistically induce activation of calcium flux and NFAT signaling pathways, regulating the expression of cytokines including IL-2, IL-4, IL-5 and IFN- ⁇ [12] .
  • OX40 activation can regulate T cell proliferation, apoptosis and cytokine secretion activity through NF- ⁇ B signaling pathway, PKB/PI3K signaling pathway and NFAT signaling pathway, thereby enhancing the immune system viability.
  • OX40 costimulatory signal can achieve immune activation by enhancing the effector T cell activity and inhibiting Treg function in some cases; on the other hand, OX40 signaling pathway may also promote Treg cell proliferation, antagonistic effect T In addition, the mechanism of action of ADCC can both remove Teff cells and clear negatively regulated Treg cells.
  • the present invention provides novel anti-human OX40 monoclonal antibodies, polynucleotides encoding the same and their use in tumor therapy.
  • the heavy chain complementarity determining region is: SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3; SEQ ID NO: 1, SEQ ID NO: 7 and/or SEQ ID NO: 3. Or SEQ ID NO: 9, SEQ ID NO: 10 and/or SEQ ID NO: 11.
  • the light chain complementarity determining region is: SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6; SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 8. Or SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • the heavy chain complementarity determining region comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain complementarity determining region comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;
  • the heavy chain complementarity determining region comprises SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3, and the light chain complementarity determining region comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;
  • the complementarity determining region comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain complementarity determining region comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8;
  • heavy chain complementation determining The region includes SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3, and the light chain complementarity determining region comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8; or a heavy chain complementarity determining region
  • heavy chain variable region is replaced by the sequence: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 29.
  • light chain variable region is replaced by the sequence: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof is a humanized or fully human monoclonal antibody.
  • the antibody or antigen-binding fragment thereof is a camelized single domain antibody, a bifunctional antibody, a scFv, a scFv dimer, a BsFv, a dsFv, a dsFv2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab) ') 2, ds bifunctional antibody, Nanobody, domain antibody or bivalent domain antibody.
  • immunoglobulin constant region including a constant region of a protein of human IgG1, IgG2 or IgG4.
  • the antibody or antigen-binding fragment thereof provided by the present invention further comprises a conjugate.
  • the invention provides an isolated polynucleotide encoding according to the antibody or antigen-binding fragment thereof.
  • the invention provides a vector comprising the isolated polynucleotide.
  • the invention provides a host cell comprising the vector.
  • the invention provides a method of expressing the antibody or antigen-binding fragment thereof, comprising the host cell cultured under conditions in which the isolated polynucleotide is expressed.
  • the invention also provides a kit comprising the antibody or antigen-binding fragment thereof.
  • the invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.
  • the invention provides the use of the antibody or antigen-binding fragment thereof for detecting the presence or level of human or monkey OX40.
  • the invention provides the use of the antibody or antigen-binding fragment thereof for detecting an individual having a condition or condition responsive to an OX40 agonist.
  • the invention provides the use of the antibody or antigen-binding fragment thereof for monitoring the therapeutic response or progression of disease treated with an OX40 agonist.
  • the invention provides the use of the antibody or antigen-binding fragment thereof for the manufacture of a medicament for the treatment of a condition which would benefit from an up-regulated immune response.
  • the condition is cancer or a chronic viral infection.
  • the invention provides a method of detecting the presence or level of human or monkey OX40, the method comprising the use of an antibody of the invention or an antigen binding fragment thereof.
  • the invention provides a method of detecting a condition or condition that is responsive to an OX40 agonist, the method comprising using an antibody of the invention or an antigen binding fragment thereof.
  • the invention provides a method of monitoring a therapeutic response or progression of a disease treated with an OX40 agonist, the method comprising using an antibody of the invention or an antigen binding fragment thereof.
  • the invention provides a method of treating a condition that would benefit from an up-regulated immune response, the method comprising using an antibody of the invention or an antigen-binding fragment thereof.
  • the condition is cancer or a chronic viral infection.
  • the present invention provides an antibody or antigen-binding fragment thereof for detecting the presence or level of human or monkey OX40.
  • the invention provides an antibody or antigen-binding fragment thereof for detecting a condition or condition that is responsive to an OX40 agonist.
  • the invention provides an antibody or antigen-binding fragment thereof that monitors the therapeutic response or progression of disease treated with an OX40 agonist.
  • the invention provides an antibody or antigen-binding fragment thereof that treats a condition that would benefit from an up-regulated immune response.
  • the condition is cancer or a chronic viral infection.
  • the beneficial effects of the invention the invention develops a human-mouse chimeric anti-OX40 monoclonal antibody by modifying the self-developed murine anti-OX40 antibody, and the antibody of the invention can bind to the cell surface OX40 Proteins and activate their downstream signaling pathways, which activate the function of T cells, thus providing a possibility to treat tumors or chronic viral infections.
  • Figure 1 shows the results of an ELISA binding assay.
  • Figure 2 shows the results of a binding assay of FACS detection antibody to CHO-hsOX40.
  • Figure 3 shows the results of a binding assay of FACS detection antibody to CHO-mOX40.
  • Figure 4 shows that OX40 antibody activates NF-kB signaling pathway activity in Jurkat cells.
  • Figure 5 shows that OX40 antibody competitively inhibits OX-40L-induced activation of the NF-kB signaling pathway in Jurkat cells.
  • Figure 6 shows that OX40 antibody mediates the ADCC effect of PBMC on CHO-OX40 cells.
  • FIG. 7 shows that OX40 antibody promotes proliferation of Th cells. Different concentrations of OX40 antibody synergize with OKT3 to promote proliferation of CD4+ Th cells.
  • Figure 8a shows the relationship between OX40 antibody drug inhibition of tumor volume (**P ⁇ 0.01), and Figure 8b shows that OX40 antibody drug has no significant effect on mouse body weight.
  • Figure 9 shows ELISA data for binding of humanized antibody (a) to human OX40; (b) activation activity of NF-kB signaling pathway in Jurkat cells; and (c) anti-tumor drugs on OX40 humanized transgenic mice effect.
  • Figure 10a shows the pharmacokinetic profile of OX40 antibody drug in mice
  • Figure 10b shows the pharmacokinetic profile of OX40 antibody drug in cynomolgus monkeys.
  • antibody includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody which binds to a specific antigen.
  • a natural intact antibody contains two heavy chains and two light chains. Each heavy chain is composed of a variable region and first, second, and third constant regions; each light chain is composed of a variable region and a constant region. Mammalian heavy chains can be divided into ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , and mammalian light chains can be divided into ⁇ or ⁇ .
  • the antibody is of the "Y" type and the neck of the Y-form consists of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds.
  • Each arm of the "Y" type structure includes a variable region of one of the heavy chains and a first constant region that binds to the variable and constant regions of a light chain.
  • the variable regions of the light and heavy chains determine the binding of the antigen.
  • the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs).
  • the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3, and the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3.
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or recognized by the Kabat, Chothia or Al-Lazikani nomenclature.
  • the three CDRs are separated by a continuous side portion called a frame region (FR), which is more highly conserved than the CDR and forms a support for the hypervariable loop.
  • FR frame region
  • the constant regions of the heavy and light chains are not associated with antigen binding, but have multiple effector functions.
  • Antibodies can be classified into several classes depending on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms, depending on whether they contain alpha, delta, epsilon, gamma and mu heavy chains: IgA, IgD, IgE, IgG, and IgM.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding fragment refers to an antibody fragment formed by an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have an intact antibody structure.
  • antigen-binding fragments include, but are not limited to, such as diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2.
  • Bispecific dsFv (dsFv-dsFv'), disulfide stabilized bifunctional antibody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single chain antibody (BsFv), multispecific antibodies, camelized single domain antibodies, Nanobodies, domain antibodies, and bivalent domain antibodies.
  • the antigen binding fragment can bind to the same antigen as the parent antibody.
  • an antigen-binding fragment may contain one or more CDRs from a particular human antibody, ligated to a framework region from one or more different human antibodies.
  • a “Fab” fragment of an antibody refers to the portion of the antibody molecule that is bound by a disulfide bond between a light chain (including the variable region and the constant region) and a variable region and a partial constant region of a heavy chain.
  • a “Fab” fragment refers to a Fab fragment comprising a portion of the hinge region.
  • F(ab')2 refers to the dimer of Fab.
  • the Fc portion of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but does not participate in antigen binding.
  • the "Fv” segment of an antibody refers to the smallest antibody fragment that contains the entire antigen binding site.
  • the Fv fragment consists of a variable region of a light chain and a variable region of a heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody that is directly linked by a light chain variable region to a heavy chain variable region or joined by a peptide chain (Huston JS et al, Proc Natl Acad Sci USA, 85: 5879 (1988)).
  • Single-chain antibody Fv-Fc or “scFv-Fc” refers to an engineered antibody consisting of an scFv linked to the Fc portion of an antibody.
  • “Camelized single domain antibody, “heavy-chain-only antibodies” or “HCAb” refers to antibodies that contain two VH domains but no light chain (Riechmann L And Muyldermans S., J Immunol Methods. 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. 74(4): 277-302 (2001); W094/04678; W094/25591 US Patent No. 6,005,079. Heavy chain antibodies were originally derived from camelids (camel, dromedary, and llama).
  • variable region of the heavy chain antibody (VH domain) is the smallest known antigen-binding unit for acquired immunity (Koch-Nolte F. et al., FASEB J. 21(13): 3490-8. Epub (2007)) .
  • Nanobody refers to an antibody fragment consisting of a VH domain from a heavy chain antibody and two constant regions, CH2 and CH3.
  • a “diabody” includes a small antibody fragment with two antigen binding sites, wherein the fragment contains a VH domain and a VL domain linked together on the same polypeptide chain (see, Holliger P. et al., Proc Natl). Acad Sci U S A.90(14):6444-8 (1993); EP404097; W093/11161).
  • the link between the two domains is so short that the two domains on the same chain cannot be paired with each other, forcing the two domains to pair with the complementary domains of the other two strands to form two antibody binding sites.
  • These two antibody binding sites can be targeted to bind to the same or different antigens (or epitopes).
  • Domain antibody refers to an antibody fragment that contains only one heavy chain variable region or one light chain variable region.
  • two or more VH domains are covalently bound by a polypeptide linker and form a bivalent domain antibody.
  • the two VH domains of a bivalent domain antibody can target the same or different antigens.
  • "(dsFv)2" contains three peptide chains: the two VH genes are joined by a single polypeptide linker and joined to the two VL groups by a disulfide bond.
  • the "bispecific ds bifunctional antibody” comprises VL1-VH2 (linked by two polypeptide linkers) and VH1-VL2 (also linked by two polypeptide linkers), between VH1 and VLl Bonded by disulfide bonds.
  • Bispecific dsFv or "dsFv-dsFv” contains three polypeptide chains: the VH1-VH2 group, wherein the heavy chains of the two are linked by a polypeptide linker (eg, a long elastic linker) and are separated by a disulfide bond.
  • a polypeptide linker eg, a long elastic linker
  • disulfide bond In combination with the VL1 and VL2 groups, each pair of heavy chain light chains paired by a disulfide bond has a different antigen specificity.
  • the VH of the two groups cooperates with the VL of the other group to form two binding sites that can be targeted to bind to the same antigen (or antigenic epitope) or to different antigens (or epitopes).
  • the "scFv dimer" is a bispecific bifunctional antibody comprising an interconnected V L1 -V H2 (linked by a polypeptide linker) and V H1 -V L2 (attached by a polypeptide linker) Where V H1 and V L1 cooperate, V H2 and V L2 cooperate, and each cooperating pair has a different antigen specificity.
  • full human when applied to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to a human Or an amino acid sequence of an antibody produced by a human immune cell or derived from a non-human source such as a transgenic non-human animal using a human antibody library, or other sequence encoding a human antibody.
  • the fully human antibody does not comprise an amino acid residue (particularly an antigen binding residue) derived from a non-human antibody.
  • humanized when applied to an antibody or antigen-binding fragment, is meant to include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable) An antibody or antigen-binding fragment. Since humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for humans in certain embodiments.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig or hamster.
  • the humanized antibody or antigen-binding fragment consists essentially of a human sequence, except that the CDR sequences are non-human.
  • the human derived FR region can comprise the same amino acid sequence as the human antibody from which it is derived, or it can include some amino acid changes, eg, no more than 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid change.
  • the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or both.
  • the humanized antibody comprises human FR1-3 and human JH and JK.
  • chimeric refers to a portion of a heavy chain and/or a light chain derived from one species, and the remainder of the heavy and/or light chain is derived from an antibody or antigen binding of a different species. Fragment.
  • a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse.
  • OX40 refers to a receptor that binds to OX40L. It is a type I membrane protein belonging to the TNF receptor family. Other names are ACT-4, OX40L receptor, CD134 antigen, ACT35 antigen, TNFRSF4. It has a molecular weight of 50 kDa and is stored in SwissProt under accession number P43489.
  • anti-OX40 antibody refers to an antibody that is capable of specifically binding to OX40 (eg, human or monkey OX40) with sufficient affinity for diagnostic and/or therapeutic use.
  • telomere binding refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen.
  • an antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey OX40 and has a binding affinity (K D ) ⁇ 10 -6 M.
  • K D in the present application means the ratio of the dissociation speed to the bonding speed (k off / kon ), which can be determined by surface plasmon resonance, for example using an instrument such as Biacore.
  • MT01-L1 refers to a heavy chain variable region as set forth in SEQ ID NO: 15, a light chain variable region as set forth in SEQ ID NO: 16, and a human IgG1/ ⁇ homologous species. Human-murine chimeric monoclonal antibody of the constant region.
  • M1 refers to a heavy chain variable region as shown in SEQ ID NO: 17, a light chain variable region as shown in SEQ ID NO: 16, and a human IgG1/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • M2 refers to a heavy chain variable region as shown in SEQ ID NO: 15, a light chain variable region as shown in SEQ ID NO: 18, and a human IgG1/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • M1/M2 refers to a heavy chain variable region as shown in SEQ ID NO: 17, a light chain variable region as shown in SEQ ID NO: 18, and a human source.
  • MT01-L1(G2) refers to a heavy chain variable region as set forth in SEQ ID NO: 15, a light chain variable region as set forth in SEQ ID NO: 16, and a human IgG2/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • MT01-L2 refers to a heavy chain variable region as set forth in SEQ ID NO: 19, a light chain variable region as set forth in SEQ ID NO: 20, and a human IgG1/ ⁇ homologous species. Human-murine chimeric monoclonal antibody of the constant region.
  • MT01-L2(G2) refers to a heavy chain variable region as set forth in SEQ ID NO: 19, a light chain variable region as set forth in SEQ ID NO: 20, and a human IgG2/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • MMT01-C1 refers to a heavy chain variable region as set forth in SEQ ID NO: 29, a light chain variable region as set forth in SEQ ID NO: 30, and a human IgG1/ ⁇ homologous species. Humanized monoclonal antibodies of the constant region of the type.
  • MT01-C1(G2) refers to a heavy chain variable region as set forth in SEQ ID NO: 29, a light chain variable region as set forth in SEQ ID NO: 30, and a human IgG2/ A humanized monoclonal antibody to the ⁇ isoform constant region.
  • a “conservative substitution” is used in the present application for an amino acid sequence, it is meant that one amino acid residue is replaced with another amino acid residue having a side chain having similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (eg, Met, Ala, VaL, Leu, and Ile), neutral hydrophilic side chain residues (eg, Cys, Ser, Thr, Asn, and Gln), acidic side chain residues Conservative substitutions between the bases (eg, Asp, Glu), between basic side chain amino acids (eg, His, Lys, and Arg) or between aromatic side chain residues (eg, Trp, Tyr, and Phe). It is well known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of the protein and thus retain the biological activity of the protein.
  • hydrophobic side chain amino acid residues eg, Met, Ala, VaL, Leu, and Ile
  • neutral hydrophilic side chain residues eg, Cys, Ser,
  • percent sequence identity when used for an amino acid sequence (or nucleic acid sequence), it means that the sequence alignment is performed, and if necessary, the interval is introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence,
  • the amino acid (or nucleic acid) residue that is identical to the sequence occupies a percentage of the amino acid (or nucleic acid) residue of the candidate sequence. A conservative substitution of the amino acid residue may or may not be considered to be the same residue.
  • Sequences can be aligned by means of the tools disclosed in the art to determine the percent sequence identity of an amino acid (or nucleic acid) sequence. Those skilled in the art can adjust the parameters using the default parameters of the tool or according to the needs of the alignment, for example by selecting a suitable algorithm.
  • T cells include CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.
  • effector function refers to the biological activity of the Fc region of an antibody that binds to its effectors, such as the C1 complex and the Fc receptor.
  • exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by C1q interaction between an antibody and a C1 complex, antibody-dependent cell-mediated cells induced by binding of an Fc region of an antibody to an Fc receptor on an effector cell Toxicity (ADCC) and phagocytosis.
  • CDC complement-dependent cytotoxicity
  • ADCC effector cell Toxicity
  • cancer or “cancer condition” refers to any medical condition mediated by growth, proliferation or metastasis of a tumor or malignant cell and which elicits a solid tumor and a non-solid tumor such as leukemia.
  • tumor in the present invention means a solid substance of a tumor and/or a malignant cell.
  • Treatment of a condition includes preventing or mitigating a condition, reducing the rate at which a condition arises or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition. , reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, healing a condition, or a combination of the above.
  • Treatment or “therapy” for cancer can refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination of the above.
  • Treatment or “therapy” for a tumor includes clearance of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor progression, or some combination of the above.
  • the “separated” substance has been artificially altered by the natural state. If a “separated” substance or component appears in nature, it has been altered or removed from its original state, or both.
  • a polynucleotide or polypeptide naturally occurring in a living animal is not isolated, but if the polynucleotide or polypeptide is sufficiently separated from the substance in its natural state and exists in a sufficiently pure state, I think it is "separated.”
  • the antibody and antigen-binding fragment are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure by electrophoretic methods (eg, SDS-PAGE, isoelectric focusing) , capillary electrophoresis), or chromatography (such as ion exchange chromatography or reversed phase HPLC).
  • the "vector” in the present invention means a vehicle which can operably insert a polynucleotide encoding a protein into it and obtain expression of the protein.
  • the vector can be used to transform, transduce or transfect a host cell such that the genetic material elements carried thereby are expressed in the host cell.
  • the vector includes: a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC), a phage such as a ⁇ phage or M13 phage, as well as animal viruses and the like.
  • the animal viruses used as vectors are retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses ( Such as SV40)).
  • the vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • the vector may also include components that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein shells.
  • a "host cell” in the present invention is a cell that directs into an exogenous polynucleotide and/or vector.
  • the "disease associated with or associated with OX40" in the present invention means any condition which is caused, aggravated or otherwise related due to an increase or decrease in OX40 expression or activity.
  • a therapeutically effective amount or “effective amount” in the present invention is meant a dose or concentration of a medicament effective to treat a disease or condition associated with human OX40.
  • a therapeutically effective amount is that at that dose or concentration, the antibody or antigen conjugate can eliminate all or part of the tumor, inhibit or slow tumor growth, inhibit Mediating the growth or reproduction of cells in a cancerous state, inhibiting tumor cell metastasis, alleviating any symptoms or markers associated with a tumor or cancer condition, preventing or delaying the progression of a tumor or cancer condition, or some combination of the above.
  • “Pharmaceutically acceptable” means the carrier, vehicle, diluent, excipient, and/or salt referred to, and is generally chemically and/or physically compatible with the other ingredients of the formulation, and Physically compatible with the recipient.
  • the present application provides exemplary monoclonal antibodies MT01-L1, MT01-L1 (M1), MT01-L1 (M2), MT01-L1 (M1/M2), MT01-L1 (G2) , MT01-L2 and MT01-L2 (G2), MT01-C1 and MT01-C1 (G2).
  • M1 MT01-L1
  • M2 MT01-L1
  • M1/M2 MT01-L1
  • G2 MT01-L2 and MT01-L2
  • G2 MT01-C1 and MT01-C1
  • G2 MT01-C1 and MT01-C1
  • the anti-OX40 antibody and antigen binding fragment thereof comprise a sequence of heavy chain complementarity determining regions selected from the group consisting of SEQ ID NOs: 1, 2, 3, 7, 9, 10, and 11. In some embodiments, the anti-OX40 antibody and antigen binding fragment thereof comprise a sequence of a light chain complementarity determining region selected from the group consisting of SEQ ID NOs: 4, 5, 6, 8, 12, 13 and 14.
  • the anti-OX40 antibody and antigen binding fragment thereof comprise a light chain complementarity determining region selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO:6; a light chain complementarity determining region comprising SEQ ID NO:4, SEQ ID NO:5 and/or SEQ ID NO:8; and a light chain complementarity determining region comprising SEQ ID NO:12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • MT01-L1 refers to a heavy chain variable region as set forth in SEQ ID NO: 15, a light chain variable region as set forth in SEQ ID NO: 16, and a human IgG1/ ⁇ homologous species. Human-murine chimeric monoclonal antibody of the constant region.
  • M1 refers to a heavy chain variable region as shown in SEQ ID NO: 17, a light chain variable region as shown in SEQ ID NO: 16, and a human IgG1/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • MT01-L1(G2) refers to a heavy chain variable region as set forth in SEQ ID NO: 15, a light chain variable region as set forth in SEQ ID NO: 16, and a human IgG2/ Human-mouse chimeric monoclonal antibody to the ⁇ isoform constant region.
  • MT01-L2 refers to a heavy chain variable region as set forth in SEQ ID NO: 19, a light chain variable region as set forth in SEQ ID NO: 20, and a human IgG1/ ⁇ homologous species. Human-murine chimeric monoclonal antibody of the constant region.
  • CDR sequences can be modified to include substitutions of one or more amino acids, thereby resulting in increased biological activity such as increased binding affinity to human OX40.
  • phage display technology can be used to produce and express a library of antibody variants (eg, Fab or FcFv variants) followed by screening for antibodies that have affinity for human OX40.
  • computer software can be used to mimic the binding of the antibody to human OX40 and to identify amino acid residues on the antibody that form a binding interface. Substitution of these residues can be avoided to prevent a decrease in binding affinity, or these residues can be targeted for substitution to form a stronger binding.
  • at least one (or all) of the substitutions in the CDR sequences are conservative substitutions.
  • the antibodies and antigen-binding fragments thereof of the present application bind to human OX40 at an EC50 (ie, 50% effective concentration) of 0.1 ⁇ g/mL to 10 ⁇ g/mL (as determined using FACS analysis).
  • the anti-OX40 antibody and antigen-binding fragment thereof described in the present application may be a monoclonal antibody, a polyclonal antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, and a bivalent antibody.
  • Recombinant antibodies are antibodies that are produced in vitro using recombinant methods rather than animals.
  • a bispecific antibody or bivalent antibody is an artificial antibody having a fragment of two different monoclonal antibodies that binds two different antigens.
  • a "bivalent" antibody and its antigen-binding fragment include two antigen-binding sites. The two antigen binding sites may bind to the same antigen or may each bind to a different antigen, in which case the antibody or antigen binding fragment is "bispecific".
  • an anti-OX40 antibody and antigen-binding fragment thereof described herein further comprise an immunoglobulin constant region.
  • the immunoglobulin constant region comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises a CH1, CH1-CH2 or CH1-CH3 region.
  • the immunoglobulin constant region can further comprise one or more modifications to achieve the desired properties. For example, the constant region can be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.
  • the anti-OX40 antibody and antigen-binding fragment thereof further comprise a conjugate.
  • the antibodies or antigen-binding fragments thereof of the invention can be linked to a variety of conjugates (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr. (eds.), Carger Press). New York (1989)). These conjugates can be linked to the antibody or antigen conjugate by covalent attachment, affinity binding, insertion, association binding, complexation, binding, mixing or addition.
  • detectable labels may include fluorescent labels (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (eg, horseradish peroxidase, alkaline phosphatase) , luciferase, glucoamylase, lysozyme, sugar oxidase or ⁇ -D galactosidase), stable isotope or radioisotope, chromophore moiety, digoxin, biotin/avidin, DNA molecule Or gold for testing.
  • the conjugate can be a pharmacokinetic modifying moiety such as PEG that helps to increase the half-life of the antibody.
  • the conjugate can be a purification moiety such as a magnetic bead.
  • a "cytotoxic moiety" can be any agent that is harmful to the cell or that can damage or kill the cell.
  • cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, brominated bromide, ipecaine, mitomycin, etopox, teniposide, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoid, Procaine, tetracaine, lidocaine, procatrodol, puromycin and its analogues, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-ostrich, arsenic Cytosine, 5-fluorouracil dacaba), alkylating agents (eg nitrogen mustard, thiotebutazone, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclo
  • a vector comprising a polynucleotide encoding the anti-OX40 antibody and its antigen-binding fragment can be introduced into a host cell for cloning (amplification of DNA) or using recombinant techniques well known in the art. gene expression.
  • the antibody can be made by methods of homologous recombination well known in the art.
  • the DNA encoding the monoclonal antibody can be isolated and sequenced by conventional methods (e.g., an oligonucleotide probe can be used which specifically binds to the gene encoding the heavy and light chains of the antibody).
  • a variety of carriers are available.
  • Vector components typically include, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (eg, SV40, CMV, EF-1a) and Transcription termination sequence.
  • a signal sequence typically includes, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (eg, SV40, CMV, EF-1a) and Transcription termination sequence.
  • the vector system comprises a mammalian, bacterial, yeast system, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2 Other commercially available or commercially available vectors such as pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, and the like.
  • Suitable vectors can include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).
  • a vector comprising a polynucleotide encoding the antibody and antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression.
  • the host cell suitable for cloning or expressing the DNA in the vector in the present invention is a prokaryotic cell, a yeast or the above-described high-grade eukaryotic cell.
  • Prokaryotic cells suitable for use in the present invention include eubacteria such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella Genus, Proteus, Salmonella, such as, Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and Bacillus, Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas and Streptomyces.
  • Enterobacteriaceae such as Escherichia coli, Enterobacter, Erwinia, Klebsiella Genus, Proteus
  • Salmonella such as, Salmonella typhimurium
  • Serratia such as Serratia marcescens, and Shigella
  • Bacillus Bacillus subtilis and Bacillus licheniformis
  • Pseudomonas such
  • eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cell clones or to express vectors encoding anti-OX40 antibodies.
  • Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism.
  • Kluyveromyces such as Kluyveromyces cerevisiae, Kluyveromyces cerevisiae (ATCC 12, 424) ), Kluyveromyces cerevisiae (ATCC16, 045), Kluyveromyces cerevisiae (ATCC24, 178), Klewingro yeast (ATCC56,500), Kluyveromyces cerevisiae (ATCC36, 906), heat-resistant gram Rubrita yeast and K.
  • Kluyveromyces such as Kluyveromyces cerevisiae, Kluyveromyces cerevisiae (ATCC 12, 424) ), Kluyveromyces cerevisiae (ATCC16, 045), Kluyveromyces cerevisiae (ATCC24, 178), Klewingro yeast (ATCC56,500), Kluyveromyces cerevisiae (ATCC36, 906), heat-resistant gram Rubrita yeast and K.
  • marxianus Yarrowia lipolytica (EP 402, 226); Pichia pastoris (EP 183, 070); Candida: Trichoderma reesei (EP 244, 234); Western Schwann yeast, such as: Western Schwann yeast; and filamentous fungi, such as: Neurospora, Penicillium, Curvularia and Aspergillus, such as: Aspergillus niger and Aspergillus niger.
  • Host cells suitable for expressing a glycosylated antibody or antigen-binding fragment thereof, which are provided in the present invention are derived from a multicellular organism.
  • invertebrate cells include plant and insect cells.
  • a variety of baculoviral strains and variants thereof, as well as corresponding permissive insect host cells, have been discovered, from hosts such as: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito) ), Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila) and silkworm.
  • a variety of viral strains for transfection are publicly available, such as the Bm-5 variant of the genus Helicoverpa armigera nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, which can be used in the present invention, particularly For transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes, and tobacco can also be used as hosts.
  • SV40-transformed monkey kidney cell CV1 line COS-7, ATCC CRL 1651
  • human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al.,].
  • Gen Virol. 36:59 (1977) hamster kidney cells (B blood, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77 : 4216 (1980)); mouse testis-supporting cells (TM4, Ma ther, Biol. Reprod.
  • the supernatant of the expression system is typically first concentrated using a commercially available protein concentration filter, such as 1 Amicon or Millipore Pellicon ultrafiltration unit.
  • Protease inhibitors such as PMSF can be added to inhibit protein degradation, as well as antibiotics to prevent the growth of incidental contaminants in any of the foregoing steps.
  • Protein purification techniques can also be determined based on the antibodies obtained, such as fractionation in an ion exchange column, ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin agarose gel chromatography based on anion or cation exchange resin (eg poly aspartame) Column, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.
  • the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (eg, From about 0 to 0.25 M salt concentration).
  • the kit comprises an anti-OX40 antibody conjugated to a detectable label and an antigen binding fragment thereof. In some embodiments, the kit comprises an unlabeled anti-OX40 antibody and an antigen binding fragment thereof, and further comprising a secondary antibody capable of binding to an unlabeled anti-OX40 antibody and an antigen binding fragment thereof.
  • the kit may further comprise instructions for use and a package that separates each component in the kit.
  • the anti-OX40 antibody and antigen-binding fragment thereof are ligated to a substrate or instrument for use in a sandwich assay such as ELISA or immunochromatographic assay.
  • a substrate or instrument for use in a sandwich assay such as ELISA or immunochromatographic assay.
  • Suitable substrates or instruments can be, for example, microplates and test strips.
  • the application further provides a pharmaceutical composition comprising the anti-OX40 antibody and an antigen binding fragment thereof, and one or more pharmaceutically acceptable carriers.
  • one or more antioxidants such as methionine
  • a composition comprising an antibody or antigen-binding fragment thereof disclosed herein, which may reduce the antibody or antigen-binding fragment thereof Oxidation.
  • a reduction in oxidation prevents or reduces the decrease in binding affinity, thereby increasing antibody stability and extending shelf life.
  • the compositions provided herein comprise one or more of said antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine.
  • the present invention further provides methods for preventing oxidation of the antibody or antigen-binding fragment thereof by mixing the antibody or antigen-binding fragment thereof provided in the present invention with one or more antioxidants, such as methionine. , extend its shelf life and / or increase its activity.
  • the pharmaceutically acceptable carrier can include, for example, an aqueous medium such as sodium chloride injection, Ringer's solution, isotonic glucose injection, sterile water injection, or glucose and lactate.
  • aqueous medium such as: plant-derived fixed oil, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at the concentration of bacterial inhibition or fungal inhibition, isotonic agents such as: sodium chloride or glucose , buffer such as: phosphate or citrate buffer, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethyl cellulose, hydroxypropyl Methyl cellulose or polyvinylpyrrolidone, emulsifier such as polysorbate 80 (Tween-80), integration reagent such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl) Ether
  • the antibacterial agent as a carrier can be added to the pharmaceutical composition in a multi-dose container, including phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thiosemeril, chlorobenzylammonium chloride and chlorophenylethylammonium.
  • Suitable excipients can include, for example, water, salt, glucose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrin. substance.
  • the unit dose of the injectable preparation is packaged in an ampoule, a tube or a syringe with a needle. It is well known in the art that all formulations for administration by injection should be sterile and pyrogen free.
  • filtration sterilization of the solution is carried out under standard conditions well known in the art and then lyophilized to give the desired formulation.
  • the resulting solvent is dispensed into a vial and lyophilized.
  • Each tubule can hold a single dose or multiple doses of the anti-OX40 antibody or antigen-binding fragment thereof, or a combination thereof.
  • the amount of loading in each vial may be slightly higher than required for each dose or multiple doses (eg, 10% excess) to ensure accurate sampling and precise administration.
  • the lyophilized powder can be stored under suitable conditions, such as in the range of from about 4 ° C to room temperature.
  • Also provided are methods of treatment comprising administering a therapeutically effective amount of an anti-OX40 antibody or antigen-binding fragment thereof described herein to a subject in need thereof, thereby treating or preventing a condition or disorder associated with OX40.
  • a method of treating a condition of a subject that would benefit from an up-regulated immune response comprising administering to the subject in need thereof a therapeutically effective amount of an anti-OX40 antibody described herein or Its antigen-binding fragment.
  • an anti-OX40 antibody or antigen-binding fragment thereof provided herein is dependent on a variety of factors well known in the art, such as body weight, age, past medical history, current treatment, the health of the subject, and the potential for cross-infection, allergies. Hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. A person skilled in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage according to these or other conditions or requirements.
  • an anti-OX40 antibody or antigen-binding fragment thereof provided herein can be administered at a therapeutically effective dose of between about 0.01 mg/kg to about 100 mg/kg.
  • the anti-OX40 antibody or antigen-binding fragment thereof is administered at a dose of about 50 mg/kg or less, and in certain embodiments, at a dose of 10 mg/kg or less, 5 mg/ Kg or less, 1 mg/kg or less, 0.5 mg/kg or less or 0.1 mg/kg or less.
  • a particular dose can be administered at multiple intervals, such as once a day, twice daily or more, twice or more per month, once a week, once every two weeks, once every three weeks, once a month, or every two Month or more.
  • the dosage administered can vary with the course of the treatment. For example, in certain embodiments, the initial dose administered can be higher than the subsequent dose. In certain embodiments, the administered dose is adjusted during the course of treatment depending on the response of the subject to be administered.
  • the antibodies and antigen-binding fragments disclosed in the present invention can be administered by administration methods well known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular or intradermal injection).
  • injection e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular or intradermal injection.
  • non-injectable administration eg, oral administration, nasal administration, sublingual administration, rectal administration, or topical administration.
  • the conditions and conditions associated with OX40 can be immune-related diseases or conditions.
  • the conditions and conditions associated with OX40 include tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, Bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, grass granulation Mycoses fungoids, Merkel cell carcinoma and other hematological malignancies, such as classic Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/tissue cells, B-rich lymphocytes Tumor, EBV-positive and negative PTLD and EBV-related diffuse large B-cell lymphoma (DL
  • the tumor and cancer are metastatic, particularly metastatic tumors that express OX40.
  • the conditions and conditions associated with OX40 include chronic viral infections, such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I , herpes simplex virus type 2, human papilloma virus, adenovirus virus infection, Kaposi's sarcoma-associated herpes virus epidemic, Torquetenovirus, JC virus or BK virus.
  • the application further provides methods of using the anti-OX40 antibodies or antigen-binding fragments thereof.
  • the application provides a method of treating a condition or disorder associated with OX40 in an individual comprising administering a therapeutically effective amount of an anti-OX40 antibody or antigen-binding fragment thereof described herein.
  • the individual is identified as having a condition or condition that may be responsive to an OX40 agonist.
  • upregulating refers to the total level of OX40 protein detected in a sample to be tested using the antibodies or antigen-binding fragments thereof described herein, as compared to the level of OX40 protein in a reference sample detected using the same antibody. Increase by not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more many.
  • the reference sample may be a control sample obtained from a healthy or disease-free individual, or a healthy or disease-free sample obtained from an individual from which the sample is to be tested.
  • the reference sample can be a disease-free sample near or adjacent to a sample to be tested (eg, a tumor).
  • the antibodies and antigen-binding fragments disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances.
  • the antibodies and antigen-binding fragments disclosed herein can be used in combination with chemotherapy, radiation therapy, cancer treatment surgery (eg, tumor resection), one or more anti-emetic drugs or other chemotherapy-induced complications, or any other A therapeutic substance for cancer or any therapeutic substance that is mediated by OX40 is used in combination.
  • the antibodies and antigen-binding fragments disclosed herein, when used in combination with one or more therapeutic substances can be administered simultaneously with the one or more therapeutic substances, in some such
  • the antibody and antigen-binding fragment can be administered simultaneously as part of the same pharmaceutical composition.
  • antibodies and antigen conjugates that are "in combination with” other therapeutic substances need not be administered simultaneously or in the same composition as the therapeutic substance.
  • the term “combination” in the context of the present invention also includes that antibodies and antigen conjugates administered before or after another therapeutic substance are also considered to be “in combination” with the therapeutic substance, even if the antibody or antigen-binding fragment thereof The second substance is administered by different modes of administration.
  • the therapeutic substance is capable of inducing or enhancing an immune response against cancer.
  • tumor vaccines can be used to induce an immune response to certain tumors or cancers.
  • Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system.
  • cytokine treatment examples include, but are not limited to, interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as 1L-L, 1L-1a , 1L-2, 1L-3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-ll and 1L-12, tumor necrosis factor such as TNF - ⁇ and TNF- ⁇ .
  • interferons such as interferon alpha, beta and gamma
  • colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF
  • interleukins such as 1L-L, 1L-1a , 1L-2, 1L-3, 1L-4, 1L-5, 1L-6, 1L-7, 1L-8, 1L-9, 1L-10, 1L-ll and 1L
  • the present application further provides methods of monitoring a therapeutic response or disease progression in a subject treated with an OX40 agonist, comprising using the anti-OX40 antibody or antigen-binding fragment thereof described herein in a biological sample to be tested from the individual Determine the presence or level of OX40.
  • the method further comprises comparing the OX40 level in the biological sample to be tested to an OX40 level in a comparable sample previously obtained from the same individual, wherein the increase in OX40 level is decreased in the test biological sample or Slowing or stopping, indicating a positive therapeutic response or controlled disease progression.
  • the comparable sample may be the same type of sample as the sample to be tested, but it is obtained from the same individual prior to treatment or at an early stage of treatment.
  • Example 1 Obtainment of anti-human OX40 activating monoclonal antibody
  • the light chain complementarity determining region is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO: One or more of 14.
  • the heavy chain complementarity determining region is: SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3; SEQ ID NO: 1, SEQ ID NO: 7 and/or SEQ ID NO: 3; or SEQ ID NO: 9, SEQ ID NO: 10 and/or SEQ ID NO: 11.
  • the light chain complementarity determining region is: SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6; SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 8; SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • the heavy chain complementarity determining region and the light chain complementarity determining region are respectively shown in Table 1.
  • the heavy chain complementarity determining region comprises SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3; the light chain complementarity determining region comprises: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: ;
  • the heavy chain complementarity determining region comprises SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3; the light chain complementarity determining region comprises: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8. Or the heavy chain complementarity determining region comprises SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; the light chain complementarity determining region comprises: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. .
  • the inventors of the present Example 1 constructed a CHO cell line overexpressing OX40, thereby immunizing mice, and performing hybridoma cell fusion with the SP2/0-AG14 cell line.
  • a series of murine antibodies with strong ELISA binding activity and OX40 activation function were obtained by antigen-antibody binding assay and cell function screening.
  • the sequences of the variable regions VH and VL of each antibody were obtained by sequencing, and human-mouse chimeric antibodies, including MT01-L1 and MT01-L2, were designed and expressed accordingly.
  • mutations were made against potential instability specific sites in the heavy and light chain V regions.
  • the 57th amino acid Glycin of the heavy chain of MT01-L1 is mutated to Alanine
  • the 96th amino acid Tryptophan of the light chain of MT01-L1 is mutated to phenylalanine.
  • the resulting mutant antibodies were MT01-L1 (M1), MT01-L1 (M2), MT01-L1 (M1/M2).
  • a protein solution having a concentration of 1 ⁇ g/mL was coated with a 96-well high-affinity plate at 100 ⁇ L/well, and shaken overnight at 4 °C.
  • the next day the cells were washed three times with 300 ⁇ L of PBST (Tween 20: 0.5 Torr), and then blocked with 100 ⁇ L/well of 5% BSA/PBS for 2 hours, and shaken at room temperature. Wash 3 times with 300 ⁇ L PBST.
  • a gradient dilution of the sample was prepared in PBS. The plate was added to a 96-well plate at 100 ⁇ L/well, and shaken at room temperature for 1 hour. Wash 3 times with 300 ⁇ L PBST.
  • the ELISA binding EC50 of the human-mouse chimeric antibodies MT01-L1 and MT01-L2 were 290.7 ng/mL and 208.5 ng/mL, respectively.
  • the OX40 antibody gradient was configured with PBS to prepare a final concentration of 2 x working solution and operated on ice.
  • Jurkat-NFkB-luc-OX40 cells and HEK293 cells overexpressing FcR were collected, resuspended in culture medium after centrifugation.
  • OX40Ab and an appropriate amount of cell suspension were added. After incubation for 5 hours, One-Glo (Promega) detection reagent was added, and after mixing, the fluorescent signal was detected by Pherastar.
  • the EC50 of activation of the NF ⁇ B-luciferase reporter gene in the above experimental system by MT01-L1, MT01-L1 (G2), MT01-L2, and MT01-L2 (G2) was determined to be 39.1, respectively. 14.9, 84.1 and 117.9 ng/mL.
  • the results showed that the inhibitory IC50 of MT01-L1 and MT01-L2 on OX40L-induced activation of NFkB signaling pathway in Jurkat cells were 0.686 ⁇ g/mL and 1.59 ⁇ g/mL, respectively.
  • CHO-OX40-luc was plated on a 96-well flat bottom plate and incubated overnight; the next day, OX40Ab gradient dilutions were prepared in 1640 medium and added to a 96-well flat bottom plate. After incubation with human PBMC for 48 h, the supernatant was centrifuged and detected using a Cyto-Tox Glo kit. As shown in Figure 6, the results showed that the chimeric antibodies MT01-L1 and MT01-L2 had significant ADCC effects at concentrations greater than 1 ⁇ g/mL.
  • CD4+ cells were isolated from the blood of healthy volunteers and cultured in PFA/IL-2 containing 1640 complete medium for two days.
  • OKT3 0.1 ⁇ g/well
  • the gradient diluted OX40 antibody or control IgG were coated in a high affinity 96-well round bottom plate.
  • the well plates were washed twice with PBS, and the pretreated CD4+ cells were added to the antibody-coated 96-well plates at 50,000 per well, and the cells were further cultured for 7 days, and then the cell proliferation was measured using the CCK8 kit.
  • the experimental results show that OX40 antibody can promote the proliferation of CD4 Th cells in a dose-dependent manner in the presence of OKT3 antibody.
  • the viability of CD4+ cells in the case where only the highest concentration of 1500 ng/mL of OX40 antibody was added was measured in the experiment, and the data were standardized based on this.
  • Murine colon cancer cells were inoculated subcutaneously in OX40 humanized mice (OX40 extracellular segment was replaced with human OX40 extracellular segment sequence) in an amount of 10 6 cells/head. After the tumor was grown to about 100 mm 3 , OX40 antibody or IgG1 control (10 mg/kg) was administered intravenously once every 3 days for a total of 4 doses. As shown in Fig. 8 (a) and Fig. 8 (b), MT01-L1 was extremely effective compared with the control group 13 days after the start of administration. Under the same dosage regimen, although the drug effect of MT01-L2 did not reach statistically significant difference, it also showed obvious drug efficacy trend.
  • the in vitro activity assay of the humanized antibody was carried out as described in Example 5.
  • the results showed that the activation EC50 of MT01-C1 and MT01-C1 (G2) on the Jurkat-OX40-NF ⁇ B-luciferase reporter stably transfected cell line were 0.028 and 0.0434 ⁇ g/mL, respectively (Fig. 9b).
  • the animal efficacy test of the humanized antibody was carried out as in Example 8, but the administration mode of the antibody was changed to 15 mg/kg in a single administration.
  • the results showed that after 17 days of administration, the inhibition rates of tumor volume by MT01-C1 and MT01-C1 (G2) reached 79% and 66%, respectively (Fig. 9c).
  • Example 11 Humanized antibody mouse and cynomolgus PK properties
  • mice C56BL6 mice, female, 6/group were injected intravenously per antibody at a dose of 5 mg/kg. 1h, 2h, 6h, 24h, 48h, 72h, 96h, 192h, 312h after administration, 100 ⁇ L blood samples were taken from each group of mice at each time point, and after standing at 3-4h for 4 hours, centrifuged at 5000rpm for 10min, serum was taken. Store at -80 ° C for ELISA.
  • the OD value was read with a microplate reader at a detection wavelength of 450 nm and a reference wavelength of 620 nm.
  • the concentration of the antibody to be tested in the biological sample was calculated by comparing the OD450nm reading with the sample standard curve.
  • mice Male cynomolgus monkeys were intravenously administered at a dose of 1.5 mg/kg per antibody, and a group of 3 animals. Animal group received 48h before administration, 15min, 30min, 1, 2, 6, 24, 48, 72, 96, 168, 240, 336, 504h after administration, 100ul blood samples were taken at each time point, and 4 degrees were allowed to stand 2 After -3h, centrifuge at 5000rpm for 10min, serum was taken and stored at -80 °C for ELISA.
  • the OD value was read with a microplate reader at a detection wavelength of 450 nm and a reference wavelength of 620 nm.
  • the concentration of the antibody to be tested in the biological sample was calculated by comparing the OD450nm reading with the sample standard curve.

Abstract

本发明提供了一种分离的抗体或其抗原结合片段。该分离的抗体或其抗原结合片段可用于制备***的药物。

Description

分离的抗体或其抗原结合片段及其在肿瘤治疗中的应用
本申请要求2017年12月29日提交的名称为“分离的抗体或其抗原结合片段及其在肿瘤治疗中的应用”，申请号为2017114761603的中国发明专利申请的优先权。
技术领域
本发明涉及一种抗体，尤其涉及分离的抗体或其抗原结合片段及其在肿瘤治疗中的应用。
背景技术
人OX40是一个277aa的蛋白，但由于其在N146和N160位的糖基化，其表观分子量约为50kD[1,2]。OX40是一个I型穿膜蛋白，其胞外区段可与其天然配体OX40L(CD252)结合，胞内区段则与T细胞活化的多个信号通路相偶联。
人OX40主要表达于活化的T细胞上，包括CD4、CD8、Th和Treg细胞等(reviewed in[3])。在
Figure PCTCN2018120520-appb-000001
T细胞上OX40的表达很低，但是经过抗原诱导刺激后其表达水平上调并在12h至5-6天内达到峰值。类似的，OX40L的表达也受到细胞活化状态的影响[3]。APC细胞在抗原刺激后1-3天可以检测到OX40L的表达。有趣的是，除了免疫细胞外，肌肉细胞在炎症因子的刺激下也会表达OX40L[4,5]，提示OX40L-OX40信号通路可能广泛作用于机体的炎症反应。
基于OX40/OX40L主要表达于抗原激活的T细胞的特点，开发OX40激动剂有望成为一种特异性强、副作用低的免疫疗法。
抗原依赖的OX40L/OX40共刺激分子的活化与T细胞内多个信号通路相偶联。晶体结构研究表明，OX40L与OX40的结合可诱导OX40-OX40L复合物三聚体化[6]，从而在胞内形成与受体相关分子(receptor-associated factor，TRAF)的结合位点。后者(TRAF2、5)则可进而激活NF-κB信号通路，抑制T细胞凋亡[5,7,8]。有研究发现，OX40活化可以导致Bcl-2和Bcl-xL高表达[9]，提示OX40可能通过NF-κB信号通路诱导抗凋亡蛋白表达而实现其抑制T细胞凋亡的功能。
PKB/PI3K是OX40下游的另一个重要信号通路。研究发现，一方面，T 细胞上OX40的共刺激信号是维持PKB活化的必要条件，另一方面，组成型活化的PKB可以拮抗OX40缺陷导致的T细胞中抗凋亡蛋白的下调[10]。OX40共刺激信号可以通过PKB/PI3K信号通路维持Survivin的表达[11]。
最后，T细胞上TCR和OX40的活化还可以协同引起钙流和NFAT信号通路的活化，调节包括IL-2、IL-4、IL-5和IFN-γ在内的细胞因子的表达[12]。
综上，上述研究表明OX40的活化可以通过NF-κB信号通路、PKB/PI3K信号通路和NFAT信号通路来调节T细胞的增殖、凋亡和细胞因子分泌活性，从而达到增强免疫***活力的效果。
但是对OX40的更深入的研究发现，CD4 +细胞中Treg细胞亚群也会表达OX40。OX40在小鼠Treg细胞上持续表达，而人Treg细胞则是在活化后呈上调表达[13,14]。Treg是一类对效应T细胞(Teff)有抑制作用的细胞，因此研究OX40在Treg细胞上的功能是一个十分有趣的问题。现有的证据显示OX40信号通路对于天然Treg(nTreg)的发育影响不大，而大多数研究表明其对诱导的Treg(iTreg)的发育则有较明确的抑制性调节作用(reviewed in[3,15])。
与在Treg细胞发育中结论较明确不同的是，关于OX40信号通路对Treg细胞功能的调节作用的研究则不尽一致。一些研究表明OX40信号通路可以抑制Treg细胞的免疫抑制功能[14,16-20]；而另一些则发现OX40对于Treg细胞充分发挥其免疫抑制功能是必需的[21-23]。这种不一致可能与不同实验室采用的不同实验模型和实验条件相关，也提示OX40在机体内对Treg细胞的功能可能受到不同生理、病理条件的影响。
类似的，OX40活性对Treg细胞增殖和凋亡的影响也随细胞所处的微环境不同而不同。例如在没有IFN-γ和IL-4存在[24]或是有FoxP3表达[17]的情况下，激活OX40可以明显促进Treg细胞的扩增。而在另一些情况下，则没有观察到这种OX40依赖的Treg扩增[14]。
综上，OX40对免疫***的调控作用是一个复杂的过程。一方面，OX40共刺激信号在某些情况下可以通过增强效应T细胞活性和抑制Treg功能的双重机制达到免疫激活的效果；另一方面，OX40信号通路也可能促进Treg细胞的增殖，拮抗效应T细胞的功能；此外，ADCC的作用机制既能清除Teff细胞也能清除负调控的Treg细胞。
肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TIL)是外周淋巴***迁移并滞留在肿瘤组织内部的淋巴细胞，其数量及类型往往与病人的预后相关[25,26]。许多研究表明OX40在肿瘤浸润淋巴细胞上，特别是Treg上呈高表达[27-32]，这很可能是肿瘤特异抗原激活了T细胞的一种表现。在淋 巴瘤中，由于OX40在Treg上的高度上调，OX40甚至可以作为抗原特异的Treg的生物标志物[30]。有趣的是，OX40的表达与肿瘤病人的临床预后相关。在结肠癌中，TIL、肠系膜***或肿瘤边缘的淋巴集结上OX40的高表达与较长的生存期相关[31]。类似的，在黑色素瘤病人中，TIL和肿瘤周边淋巴细胞中OX40阳性细胞的数量与肿瘤的转移和病人的生存期相关[32]。
基于上述发现，以及OX40在T细胞活化中的重要作用，OX40成为肿瘤治疗的一个重要靶点。OX40信号通路的活化可以通过使用激活的OX40抗体或是与OX40L融合蛋白结合等方式来实现。最早的使用OX40激动剂进行肿瘤治疗的尝试可以追溯到2000年，Weinberg实验室报道了腹腔给予OX40L:Ig融合蛋白可以阻止皮下移植瘤的成瘤[33]。有趣的是，治疗组小鼠对于再次接种同样的肿瘤仍然有很好的免疫力，而对接种不同的肿瘤细胞则没有类似的免疫力[33]。这个结果提示OX40激动剂在肿瘤治疗过程中可以增强肿瘤特异的记忆T细胞反应。同时，研究还表明CD4 +和CD8 +细胞对于OX40激动剂的抗肿瘤活性都是必须的[33,34]。在此之后众多的临床前研究都证明了在动物模型上单独使用OX40激动剂或是与其它肿瘤治疗手段联用可以有效抑制肿瘤生长甚至预防肿瘤复发(reviewed in[15,35])。
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发明内容
为克服现有技术中的问题，本发明提供了新的抗人OX40单克隆抗体，编码其的多核苷酸及其在肿瘤治疗中的应用。
本发明提供了一种分离的抗体或其抗原结合片段，包括重链互补决定区和轻链互补决定区，所述重链互补决定区选自以下序列：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11中的一个或多个。
其中，所述重链互补决定区为：SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；或SEQ ID NO：9、SEQ ID NO：10和/或SEQ ID NO：11。
其中，所述轻链互补决定区选自以下序列：SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14中的一个或多个。
其中，所述轻链互补决定区为：SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：6；SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8；或SEQ ID NO：12、SEQ ID NO：13和/或SEQ ID NO：14。
其中，重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3,轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3,轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3,轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3,轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；或重链互补决定区包括SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11,轻链互补决定区包括SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14。
其中，所述重链可变区替换为以下序列：SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：29。
其中，所述轻链可变区替换为以下序列：SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：30。
其中，重链可变区包括SEQ ID NO：15，轻链可变区包括SEQ ID NO：16；重链可变区包括SEQ ID NO：17，轻链可变区包括SEQ ID NO：16；重链可变区包括SEQ ID NO：15，轻链可变区包括SEQ ID NO：18；重链可变区包括SEQ ID NO：17，轻链可变区包括SEQ ID NO：18；或重链可变区包括SEQ ID NO：19，轻链可变区包括SEQ ID NO：20；或重链可变区包括SEQ ID NO：29，轻链可变区包括SEQ ID NO：30；
其中，所述的抗体或其抗原结合片段是人源化或全人源单克隆抗体。
其中，所述抗体或其抗原结合片段是骆驼化单域抗体、双功能抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体、纳米抗体、域抗体或双价域抗体。
其中，其进一步包括免疫球蛋白恒定区，包括人IgG1、IgG2或IgG4的蛋白的恒定区。
本发明提供的所述抗体或其抗原结合片段，进一步包括缀合物。
本发明提供一种分离的多核苷酸，其编码根据所述抗体或其抗原结合片段。
本发明提供一种载体，其包括所述分离的多核苷酸。
本发明提供一种宿主细胞，其包括所述载体。
本发明提供一种表达所述抗体或其抗原结合片段的方法，其包括在表达所述分离的多核苷酸的条件下培养的所述宿主细胞。
本发明还提供一种试剂盒，其包括所述抗体或其抗原结合片段。
本发明还提供一种药物组合物，包括所述抗体或其抗原结合片段以及一种或多种药学上可接受的载体。
本发明提供所述抗体或其抗原结合片段在检测人或猴OX40的存在或水平中的应用。
本发明提供所述抗体或其抗原结合片段在检测鉴别患有对OX40激动剂响应的病症或状况的个体中的应用。
本发明提供所述抗体或其抗原结合片段在监测OX40激动剂治疗的治疗反应或疾病进展中的应用。
本发明提供所述抗体或其抗原结合片段在制备用于治疗会从上调的免疫响应中获益的状况的药物中的用途。其中所述状况是癌症或慢性病毒感染。
本发明提供一种检测人或猴OX40的存在或水平的方法，所述方法包括使用本发明的抗体或其抗原结合片段。
本发明提供一种检测鉴别患有对OX40激动剂响应的病症或状况的方法，所述方法包括使用本发明的抗体或其抗原结合片段。
本发明提供一种监测OX40激动剂治疗的治疗反应或疾病进展的方法，所述方法包括使用本发明的抗体或其抗原结合片段。
本发明提供一种治疗会从上调的免疫响应中获益的状况的方法，所述方法包括使用本发明的抗体或其抗原结合片段。其中所述状况是癌症或慢性病毒感染。
本发明提供一种用于检测人或猴OX40的存在或水平的抗体或其抗原结 合片段。
本发明提供一种检测鉴别患有对OX40激动剂响应的病症或状况的抗体或其抗原结合片段。
本发明提供一种监测OX40激动剂治疗的治疗反应或疾病进展的抗体或其抗原结合片段。
本发明提供一种治疗会从上调的免疫响应中获益的状况的抗体或其抗原结合片段。其中所述状况是癌症或慢性病毒感染。与现有技术相比，本发明的有益效果：本发明通过对自主研制的鼠源抗OX40抗体进行改造，研制出人-鼠嵌合抗OX40单克隆抗体，本发明的抗体能够结合细胞表面OX40蛋白并激活其下游信号通路，激活T细胞的功能，从而为***或慢性病毒感染提供了一种可能。
附图的简要说明
图1表示ELISA结合实验结果。
图2表示FACS检测抗体与CHO-hsOX40的结合实验结果。
图3表示FACS检测抗体与CHO-mOX40的结合实验结果。
图4表示OX40抗体激活Jurkat细胞NF-kB信号通路活性。
图5表示OX40抗体竞争性抑制OX-40L诱导的Jurkat细胞NF-kB信号通路的活化。
图6表示OX40抗体介导PBMC对CHO-OX40细胞的ADCC作用。
图7表示OX40抗体促进Th细胞的增殖。不同浓度的OX40抗体与OKT3协同作用，促进CD4+Th细胞的增殖。
图8a表示OX40抗体药物抑制肿瘤体积的关系图(**P<0.01)，图8b表示OX40抗体药物对小鼠体重没有明显影响。
图9表示人源化抗体(a)与人OX40结合的ELISA数据；(b)对Jurkat细胞NF-kB信号通路的激活活性；及(c)在OX40人源化转基因小鼠上的抗肿瘤药效。
图10a表示OX40抗体药物在小鼠中的药代动力学表现；图10b表示OX40抗体药物在食蟹猴中的药代动力学表现。
实施发明的最佳方式
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式、变化和修改，应理解这样的等同实施方式包括在本发明范围内。在本申请中引用的所有文献，包括 公开出版物、专利和专利申请都通过引用的方式全文并入。
术语详述
本发明中的"抗体"一词包括任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体或双特异性(双价)抗体。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一可变区和第一、第二、第三恒定区组成；每条轻链由一可变区和一恒定区组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。抗体呈"Y"型，Y型结构的颈部由两条重链的第二和第三恒定区组成，其通过二硫键结合。"Y"型结构的每条臂包括其中一条重链的可变区和第一恒定区，其与一条轻链的可变区和恒定区结合。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)。轻链(L)的CDR包含LCDR1、LCDR2、LCDR3，重链(H)的CDR包含HCDR1，HCDR2、HCDR3。本发明中公开的抗体和抗原结合片段的CDR边界可通过Kabat，Chothia或Al-Lazikani命名法命名或识别。(AI-Lazikani,B.,Chothia,C.,Lesk,A.M.,J.Mol.Biol.,273(4)：927(1997)；Chothia,C.等，J.Mol.Biol.,186(3)：651-63(1985)；Chothia,C.and Lesk,A.M.,J.Mol.Biol.,196：901(1987)；Chothia，C.等，Nature,342(6252)：877-83(1989)；Kabat,E.A.等，National Institutes of Health,Bethesda,Md.(1991))。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异构体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
本申请中的"抗原结合片段"一词，指由含有一个或多个CDR的抗体部分或者任何其他结合抗原但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包括，但不限于，如双功能抗体(diabody)、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)、双价单链抗体(BsFv)、多特异性抗体、骆驼化单域抗体(camelized single domain antibody)、纳米抗体、域抗体和双价域抗体。抗原结合片段可以与母体抗体结合相同的抗原。在某些实施方式中，抗原结合片段可以含有来自某特定人抗体的一个或多个CDR，移接至来自一个或多个不同人抗体的框架区。
抗体的"Fab"片段是指由一条轻链(包括可变区和恒定区)和一条重链的 可变区和部分恒定区经二硫键结合起来的那部分抗体分子。
"Fab’"片段是指包含了部分绞链区的Fab片段。
"F(ab')2"指的是Fab的二聚体。
抗体的Fc段负责多种不同的效应功能如ADCC和CDC，但不参与抗原的结合。
抗体的"Fv"段指的是含有完整抗原结合位点的最小抗体片段。Fv片段由一条轻链的可变区和一条重链的可变区组成。
"单链Fv抗体"或"scFv"是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体(Huston JS等，Proc Natl Acad Sci USA,85：5879(1988))。
"单链抗体Fv-Fc"或"scFv-Fc"是指由连接到某抗体Fc段的scFv组成的工程抗体。
"骆驼化单域抗体(Camelized single domain antibody)，"重链抗体"或"HCAb(Heavy-chain-only antibodies，HCAb)"都是指含有两个VH域而不含有轻链的抗体(Riechmann L.和Muyldermans S.,J Immunol Methods.231(1-2)：25-38(1999)；Muyldermans S.,J Biotechnol.74(4)：277-302(2001)；W094/04678；W094/25591；U.S.Patent No.6,005,079)。重链抗体最初从驼科(骆驼、单峰驼和美洲驼)衍生得到。虽然缺失轻链，骆驼化抗体(camelized antibodies)有确证的抗原结合全部功能(HamersCasterman C.等，Nature 363(6428)：446-8(1993)；Nguyen VK.等，"Heavy-chain antibodies in Camelidae：a case of evolutionary innovation,Immunogenetics.54(1)：39-47(2002)；Nguyen VK.等，Immunology.109(1)：93101(2003))。重链抗体的可变区(VH域)是己知的最小的获得性免疫产生的抗原结合单位(Koch-Nolte F.等，FASEB J.21(13)：3490-8.Epub(2007))。
"纳米抗体"是指一种抗体片段，其由一个来自重链抗体的VH域和两个恒定区CH2和CH3组成。
"双功能抗体(diabody)"包括带有两个抗原结合位点的小抗体片段，其中该片段含有在同一条多肽链上相连的VH域和VL域(请参见，Holliger P.等，Proc Natl Acad Sci U S A.90(14)：6444-8(1993)；EP404097；W093/11161)。两个域之间衔接物很短，使同一条链上的两个域不能互相配对，从而迫使两个域与另二条链的互补域配对，形成两个抗体结合位点。这两个抗体结合位点可靶向结合相同或不同的抗原(或抗原表位)。
"域抗体"是指仅含有一条重链可变区或一条轻链可变区的抗体片段。在某些情况下，两个或多个VH域由一个多肽衔接物共价结合并形成双价域抗体。双价域抗体的两个VH域可靶向作用于相同或不同的抗原。
在某些实施方式中，"(dsFv)2"含有三条肽链：两个VH基因间通过一条多肽衔接物相连，并通过二硫键与两个VL基团结合。
在某些实施方式中"双特异性ds双功能抗体"含有VL1-VH2(由二个多肽衔接物相连)和VH1-VL2(也是由二个多肽衔接物相连)，两者在VH1和VLl间通过二硫键结合。
"双特异性dsFv"或"dsFv-dsFv"含有三条多肽链：VH1-VH2基团，其中两者的重链通过多肽衔接物(如：长的弹性衔接物)相连，并通过二硫键分别与VL1和VL2基团结合，每对通过二硫键配对的重链轻链具有不同的抗原特异性。
在某些实施方式中，"scFv二聚体"是双价双功能抗体或双价单链抗体(BsFv)，含有二聚化的两个VH-VL(由多肽衔接物连接)基团，其中二个基团的VH与另一个基团的VL协作形成两个结合位点，这两个结合位点可靶向结合相同抗原(或抗原表位)或不同抗原(或抗原表位)。在另一些实施方式中，"scFv二聚体"是双特异性双功能抗体，含有相互连接的V L1-V H2(由多肽衔接物连接)和V H1-V L2(由多肽衔接物连接)，其中V H1和V L1协作，V H2和V L2协作，且每个协作的配对具有不同的抗原特异性。
本申请中使用的术语"全人源"当用于抗体或抗原结合片段时，是指所述抗体或抗原结合片段具有某氨基酸序列或由所述氨基酸序列组成，所述氨基酸序列对应于由人或人免疫细胞生产的、或从例如利用人源抗体库的转基因非人动物等非人来源衍生的抗体的氨基酸序列，或者其他编码人源抗体的序列。在某些实施方式中，全人源抗体不包含来源于非人抗体的氨基酸残基(特别是抗原结合残基)。
本申请中使用的术语"人源化"当用于抗体或抗原结合片段时，是指包括来源于非人动物的CDR、来源于人的FR区，以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。由于人源化的抗体或抗原结合片段具有降低的免疫原性，其在某些实施方式中可用作人的治疗剂。在一些实施方式中，所述非人动物是哺乳动物例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。在一些实施方式中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，基本上全部由人源序列组成。在一些实施方式中，所述来源于人的FR区可以包括与其来自的人源抗体相同的氨基酸序列，或其可以包括一些氨基酸改变，例如，不超过10、9、8、7、6、5、4、3、2或1个氨基酸改变。在一些实施方式中，该氨基酸改变可以仅存在于重链FR区、仅存在于轻链FR区或同时存在于两个链中。在一些优选实施方式中，所述人源化抗体包括人源FRl-3和人源JH和JK。
本申请中使用的术语"嵌合"是指具有来源于一种物种的重链和/或轻链 的一部分，和所述重链和/或轻链其余部分来源于不同物种的抗体或抗原结合片段。在一个示例性的例子中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物例如小鼠的可变区。
术语“OX40”是指与OX40L结合的受体。它是属于TNF受体家族的I型膜蛋白。其它的命名为ACT-4，OX40L受体，CD134抗原，ACT35抗原，TNFRSF4。它具有50kDa的分子量，并且存储在SwissProt中，登记号为P43489。
本申请中使用的"抗OX40抗体"是指能够以足以提供诊断和/或治疗用途的亲和性与OX40(例如人或猴OX40)特异性结合的抗体。
本申请中的"特异性结合"或"特异性的结合"是指两分子间的非随机结合反应，如抗体和抗原间的反应。在某些实施方式中，本申请的抗体或其抗原结合片段与人和/或猴OX40特异性结合，并且其结合亲和力(K D)≤10 -6M。本申请中的K D是指解离速度与结合速度的比值(k off/k on)，可通过表面等离子共振的方法测定，例如使用如Biacore的仪器。
本申请中使用的"MT01-L1"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M1)"是指具有如SEQ ID NO：17所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M2)"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：18所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M1/M2)"是指具有如SEQ ID NO：17所示的重链可变区、如SEQ ID NO：18所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(G2)"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG2/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L2"是指具有如SEQ ID NO：19所示的重链可变区、如SEQ ID NO：20所示的轻链可变区和人源IgG1/λ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L2(G2)"是指具有如SEQ ID NO：19所示的重链可变区、如SEQ ID NO：20所示的轻链可变区和人源IgG2/λ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-C1"是指具有如SEQ ID NO：29所示的重链可变区、如SEQ ID NO：30所示的轻链可变区和人源IgG1/κ同种型恒定区的人源化单克隆抗体。
本申请中使用的"MT01-C1(G2)"是指具有如SEQ ID NO：29所示的重链可变区、如SEQ ID NO：30所示的轻链可变区和人源IgG2/κ同种型恒定区的人源化单克隆抗体。
在本申请中当"保守替代"用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在疏水侧链氨基酸残基间(例如Met、Ala、VaL、Leu和Ile)、中性亲水侧链残基间(例如Cys、Ser，Thr、Asn和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)或芳香侧链残基间(例如Trp、Tyr和Phe)进行保守替代。本领域己知保守替代通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
当"百分比序列同一性"用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。所述氨基酸残基的保守替代可以认为或可以不认为是相同残基。可以通过本领域公开的工具对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。
本申请中使用的“T细胞”包括CD4+T细胞、CD8+T细胞、T辅助1型T细胞、T辅助2型T细胞、T辅助17型T细胞和抑制性T细胞。
本申请中使用的"效应功能"是指抗体的Fc区与其效应器例如C1复合物和Fc受体结合的生物活性。示例性的效应功能包括抗体与C1复合物上的C1q相互作用诱导的补体依赖性细胞毒性(CDC)、抗体的Fc区与效应细胞上的Fc受体结合诱导的抗体依赖性细胞介导的细胞毒性(ADCC)以及吞噬。
本申请中的"癌症"或"癌状况"是指任何由肿瘤或恶性细胞生长、增殖或转移所介导，并引发实体瘤和非实体瘤如白血病的医学状况。本发明中的"肿瘤"是指肿瘤和/或恶性细胞的实体物质。
对某种状况的"治疗"或"疗法"包括预防或减轻某种状况，降低某种状况兴起或发展的速度，减少发展出某种状况的风险，预防或延迟与某种状况相关的症状发展，减少或终止与某种状况相关的症状，产生某种状况的完全或部分的逆转，治愈某种状况，或以上的组合。对于癌症来说"治疗"或"疗法"可以指抑制或减缓肿瘤或恶性细胞生长，繁殖，或转移，或以上的某些组合。对于肿瘤来说"治疗"或"疗法"包括清除全部或部分的肿瘤，抑制或减缓肿瘤 生长和转移，预防或延缓肿瘤的发展，或以上的某些组合。
"被分离"的物质已经经人工由自然状态改变。如果自然界中出现某种"被分离"的物质或成分，那么其已经被改变或脱离其原始状态，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽是未被分离的，但如果这些多核苷酸或多肽与之在天然状态下共存的物质足够分离并以足够纯的状态存在，则可以认为是"被分离"。在某些实施方式中，抗体和抗原结合片段的纯度为至少90％、93％、95％、96％、97％、98％、99％，其由电泳方法(如SDS-PAGE、等电聚焦、毛细管电泳)，或色谱法(如离子交换色谱法或反相HPLC)确定。
本发明中"载体"是指，可将编码某蛋白的多核苷酸操作性地***其中并使该蛋白获得表达的一种运载工具。载体可用于转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬茵体或M13噬菌体，以及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40))。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。
本发明中"宿主细胞"是指导入外源多核苷酸和/或载体的细胞。
本发明中的"与OX40相关或有关联的疾病"是指，任何由于OX40表达或活性升高或降低而导致、加剧或其他相关的状况。
本发明中的"治疗有效量"或"有效剂量"是指，某种药物有效治疗与人OX40相关疾病或状况的剂量或浓度。例如，对于本发明中公开的抗体或其抗原结合片段的用途来说，治疗有效量是在该剂量或浓度下，该抗体或抗原结合物可以清除全部或部分肿瘤、抑制或减缓肿瘤生长、抑制介导癌状况的细胞的生长或繁殖、抑制肿瘤细胞转移、减轻任何与肿瘤或癌状况相关的症状或标记，预防或延缓肿瘤或癌状况的发展，或以上的某些组合。
"药用可接受的"是指所指的载剂、溶媒、稀释剂、辅料和/或盐，总的来说在化学上和/或在物理上与制剂中的其他配料相兼容，并在生理上与接受者相兼容。
(关于抗OX40抗体)
在某些实施方式中，本申请提供了示例性的单克隆抗体MT01-L1、 MT01-L1(M1)、MT01-L1(M2)、MT01-L1(M1/M2)、MT01-L1(G2)、MT01-L2和MT01-L2(G2)，MT01-C1和MT01-C1(G2)。其CDR序列如表1中所示，并且重链或轻链互补决定区序列也如下列出：
表1单克隆抗体序列信息
Figure PCTCN2018120520-appb-000002
在一些实施方式中，所述抗OX40抗体和其抗原结合片段包括选自下组的重链互补决定区序列：SEQ ID NO：1、2、3、7、9、10和11。在一些实施方式中，所述抗OX40抗体和其抗原结合片段包括选自下组的轻链互补决定区序列：SEQ ID NO：4、5、6、8、12、13和14。
在一些实施方式中，所述抗OX40抗体和其抗原结合片段包括选自下组的重链互补决定区：重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；以及重链互补决定区，其包括SEQ ID NO：9、SEQ ID NO：10和/或SEQ ID NO：11。
在一些实施方式中，所述抗OX40抗体和其抗原结合片段包括选自下组的轻链互补决定区：轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO： 5和/或SEQ ID NO：6；轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8；以及轻链互补决定区，其包括SEQ ID NO：12、SEQ ID NO：13和/或SEQ ID NO：14。
在一些实施方式中，所述抗OX40抗体和其抗原结合片段包括：a)重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；和轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：6；b)重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；和轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：6；c)重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；和轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8；d)重链互补决定区，其包括SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；和轻链互补决定区，其包括SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8。或；e)重链互补决定区，其包括SEQ ID NO：9、SEQ ID NO：10和/或SEQ ID NO：11；和轻链互补决定区，其包括SEQ ID NO：12、SEQ ID NO：13和/或SEQ ID NO：14。
本申请中使用的"MT01-L1"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M1)"是指具有如SEQ ID NO：17所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M2)"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：18所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(M1/M2)"是指具有如SEQ ID NO：17所示的重链可变区、如SEQ ID NO：18所示的轻链可变区和人源IgG1/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L1(G2)"是指具有如SEQ ID NO：15所示的重链可变区、如SEQ ID NO：16所示的轻链可变区和人源IgG2/κ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L2"是指具有如SEQ ID NO：19所示的重链可变区、如SEQ ID NO：20所示的轻链可变区和人源IgG1/λ同种型恒定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-L2(G2)"是指具有如SEQ ID NO：19所示的重链可变区、如SEQ ID NO：20所示的轻链可变区和人源IgG2/λ同种型恒 定区的人-鼠嵌合单克隆抗体。
本申请中使用的"MT01-C1"是指具有如SEQ ID NO：29所示的重链可变区、如SEQ ID NO：30所示的轻链可变区和人源IgG1/κ同种型恒定区的人源化单克隆抗体。
本申请中使用的"MT01-C1(G2)"是指具有如SEQ ID NO：29所示的重链可变区、如SEQ ID NO：30所示的轻链可变区和人源IgG2/κ同种型恒定区的人源化单克隆抗体。
本领域技术人员应理解可以将前述CDR序列进行修饰以包含一个或更多氨基酸的取代，由此得到提高的生物学活性例如提高的与人OX40的结合亲和性。例如，可以利用噬菌体展示技术生产并表达抗体变体库(例如Fab或FcFv变体)，随后筛选与人OX40有亲和性的抗体。另一个例子中，可以用计算机软件模拟所述抗体与人OX40的结合并鉴别抗体上形成结合界面的氨基酸残基。可以避免这些残基的替代以防止结合亲和性降低，或可以靶向这些残基进行替代以形成更强的结合。在某些实施方式中，CDR序列中的至少一个(或全部)取代是保守替代。
在某些实施方式中，所述抗体和抗原结合片段包括一个或多个CDR序列，这些序列具有与表1中所列的序列至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性，并且同时保留了与其亲本抗体相似或甚至高于其的与人OX40的结合亲和性，所述亲本抗体具有基本相同的序列，但其相应的CDR序列与表1所列的序列具有100％序列同一性。
在一些实施方式中，本申请所述抗OX40抗体和其抗原结合片段能够以≤10 -7M的结合亲和性(Kd)与人OX40特异性结合，其通过表面等离子共振法测量。结合亲和性值可以用K D值表示，其通过当抗原和抗原结合分子的结合达到平衡时的解离速率与结合速率的比值(k off/k on)计算得到。所述抗原结合亲和性(例如K D)可以通过本领域己知的适宜方法适宜地确定，例如包括使用仪器如Biacore的等离子共振结合法。
在某些实施方式中，本申请所述抗体和其抗原结合片段与人OX40以10ng/mL-10μg/mL的EC50(即半数结合浓度)结合。所述抗体与人OX40的结合可以通过本领域己知的方法如夹心法如ELISA，Western印迹，FACS或其他结合试验测定。在示例性的例子中，将待测抗体(即一抗)与固定化的人OX40或表达人OX40的细胞结合，随后洗掉未结合抗体，引入标记的二抗，其能够与一抗结合因此能够检测出结合的二抗。当使用固定化的OX40时可在酶标仪板上进行所述检测，或当使用表达人OX40的细胞时可使用FACS分析进行所述检测。
在某些实施方式中，本申请所述抗体和其抗原结合片段以0.1μg/mL至10μg/mL(使用FACS分析测定)的EC50(即50％的有效浓度)与人OX40结合。
在某些实施方式中，本申请所述抗体和其抗原结合片段可以激活人OX40信号通路，并由此提供了包括例如诱导活化的T细胞产生细胞因子(如CD4+T细胞和CD8+T细胞)、诱导活化的T细胞的增殖(如CD4+T细胞和CD8+T细胞)和逆转调节性Treg的抑制性功能的生物学活性。
所述抗OX40抗体和其抗原结合片段是人OX40特异性的。在某些实施方式中，所述抗体和其抗原结合片段不与鼠OX40结合，但与猴OX40以与人OX40相似的结合亲和性结合。例如，示例性抗体MT01-L1和MT01-L2与鼠OX40的结合用常用结合测定方法如FACS分析无法检出，而FACS检测出这些抗体与猴OX40以与人OX40相似的亲和性或EC50值结合。
在一些实施方式中，所述的抗OX40抗体和其抗原结合片段具有IgG2同种型的恒定区，其具有降低的或消除的效应功能。例如ADCC和CDC等效应功能能够导致对表达OX40的细胞的细胞毒性。一些正常的细胞能够表达OX40。为了避免对这些正常的细胞产生潜在的不希望的毒性，本发明所述的抗体和其抗原结合片段的某些实施方式具有降低的或甚至消除的效应功能。己知有许多测试用来估测ADCC或CDC活性，例如Fc受体结合试验、补体Clq结合实验和细胞裂解法，本领域技术人员能够容易选择。不希望受到理论的束缚，但据信具有降低的或消除的效应功能如ADCC和CDC的抗体不会引起对表达OX40的细胞(例如那些正常的细胞)的细胞毒性或将之降低到最小程度，因此避免了不希望的副作用。
在一些实施方式中，本申请所述的抗OX40抗体和其抗原结合片段具有降低的副作用。例如所述的抗OX40抗体和其抗原结合片段可以具有全人源IgG序列，因此其免疫原性低于人源化的抗体。再例如，所述的抗OX40抗体和其抗原结合片段可以具有IgG2或IgG4形式以消除ADCC和CDC。
在一些实施方式中，本申请所述的抗OX40抗体和其抗原结合片段的优势在于其能与具有免疫原性的物质联用，如肿瘤细胞、纯化的肿瘤抗原和用编码免疫刺激因子转染的细胞、肿瘤疫苗。此外，所述抗OX40抗体和其抗原结合片段可以包括在联用治疗中，包括标准化学疗法和放射疗法、基于靶点的小分子疗法、其他新兴免疫检查点调节剂疗法。在一些实施方式中，所述抗体和其抗原结合片段可以用作抗体一药物缀合物、双特异性或多价抗体的基础分子。
本申请所述的抗OX40抗体和其抗原结合片段可以是单克隆抗体、多克隆抗体、全人源抗体、人源化抗体、嵌合抗体、重组抗体、双特异性抗体、 标记抗体、二价抗体或抗独特型抗体。重组抗体是在体外使用重组方法而非动物制备的抗体。双特异性抗体或双价抗体是具有两种不同的单克隆抗体的片段的人工抗体，其能结合两种不同的抗原。"二价"的抗体和其抗原结合片段包括两个抗原结合位点。两个抗原结合位点可以结合相同抗原，或者可以各自结合到不同的抗原，在这种情况下，抗体或抗原结合片段为"双特异性"。
在一些实施方式中，本申请所述的抗OX40抗体和其抗原结合片段是全人源抗体。在一些实施方式中，使用重组方法制备所述全人源抗体。例如，可以制备转基因动物如小鼠，使其携带人源免疫球蛋白基因的转基因或转染色体，并因此在用适宜的抗原如人源OX40免疫后能够生产全人源抗体。全人源抗体可以从这样的转基因动物中分离，或另外地，可以通过杂交瘤技术制备，将所述转基因动物的脾细胞与永生细胞系融合以生成分泌所述全人源抗体的杂交瘤细胞。
在一些实施方式中，本申请所述的抗OX40抗体和其抗原结合片段是骆驼化单域抗体(camelized single chain domain antibody)、双功能抗体(diabody)、scFv、scFv二聚体、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体(ds diabody)、纳米抗体、域抗体或双价域抗体。
在一些实施方式中，本申请所述的抗OX40抗体和其抗原结合片段进一步包括免疫球蛋白恒定区。在一些实施方式中，免疫球蛋白恒定区包括重链和/或轻链恒定区。所述重链恒定区包括CH1、CH1-CH2或CH1-CH3区。在一些实施方式中，免疫球蛋白恒定区可以进一步包括一个或多个修饰以获得所需的性质。例如，可以将所述恒定区修饰以降低的或消除一种或多种效应功能以增强FcRn受体结合或引入一个或多个半胱氨酸残基。
在某些实施方式中，所述抗OX40抗体及其抗原结合片段进一步包含缀合物。可以设想，本发明中的抗体或其抗原结合片段可与多种缀合物连接(见例如"Conjugate Vaccines"、Contributions to Microbiology and Immunology、J.M.Cruse and R.E.Lewis、Jr.(eds.)、Carger Press、New York(1989))。这些缀合物可以通过共价结合、亲和结合、嵌入、同等结合(coordinate binding)、络合、结合、混合或加入等其他方式与所述抗体或抗原结合物连接。在某些实施方式中，本发明公开的抗体和抗原结合片段可以通过工程的方法使其含有表位结合部分以外的特定位点，这些位点可用来结合一种或多种缀合物。例如，这样的位点可包含一种或多种反应性氨基酸残基，例如半胱氨酸残基和组氨酸残基，用于协助与结合物的共价连接。在某些实施方式中，抗体可间接连于缀合物，或通过另一个缀合物相连。例如，所述抗体或其抗原结合片段可结合生物素，然后间接结合第二个缀合物，其与亲和素相连。所述缀合物可以是可检测的标记、药代动力学修饰部分、纯化部分或细胞毒性部分。 可检测的标记的例子可以包括荧光标记(例如荧光素、罗丹明、丹酰、藻红蛋白或德克萨斯红)、酶底物标记物(例如辣根过氧化物酶、碱性磷酸酶、荧光素酶、葡糖淀粉酶、溶菌酶、糖氧化酶或β-D半乳糖昔酶)、稳定同位素或者放射性同位素、发色团部分、地高辛、生物素/亲和素、DNA分子或金以进行检测。在某些实施方式中，所述缀合物可以是药代动力学修饰部分如PEG，其帮助延长抗体的半衰期。其他适宜的聚合物包括例如竣甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、乙二醇/丙二醇共聚物等。在某些实施方式中，所述缀合物可以是纯化部分例如磁珠。"细胞毒性部分"可以是对细胞有害的或可能损坏或杀死细胞的任何试剂。细胞毒性部分的示例包括，但不限于，紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙绽、吐根碱、丝裂霉素、依托泊昔、替尼泊甘、长春新碱、长春碱、秋水仙碱、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、l-去氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普茶洛尔、嘌呤霉素及其类似物、抗代谢物(例如，甲氨喋呤、6-巯基嘌呤、6-巯鸟瞟岭、阿糖胞苷、5氟尿嘧啶达卡巴)、烷化剂(例如氮芥、塞替派苯丁酸氮芥、美法仑、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲霉素、丝裂霉素C和顺-二氯二胺铂(DDP)顺铂)、蒽环类抗生素(例如柔红霉素(以前的道诺霉素)和阿霉素)、抗生素(例如更生霉素(以前称为放线菌素)、博来霉素、光神霉素和氨茵霉素(AMC))以及抗有丝***剂(例如长春新碱和长春碱)。
多核苷酸和重组方法
使用本领域公知的遗传工程学技术，可以将表1中的氨基酸序列转换成相应的DNA编码序列。由于遗传密码的简并性，转换所得的DNA序列可以完全一致，而编码的蛋白序列保持不变。
使用本领域公知的重组技术，可以将包括编码所述抗OX40抗体和其抗原结合片段(例如包括表1所示的序列)的多核苷酸的载体引入宿主细胞用于克隆(扩增DNA)或基因表达。在另一实施方式中，所述抗体可通过本领域公知的同源重组的方法制得。编码所述单克隆抗体的DNA可以通过常规的方法分离和测序(如可以使用寡核苷酸探针，该探针可特异性与编码所述抗体的重链和轻链的基因结合)。多种载体可供选择。载体组分通常包括，但不限于，以下的二种或多种：信号序列、复制起始点、一种或多种标记基因、增强序列、启动子(例如：SV40，CMV，EF-1a)和转录终止序列。
在一些实施方式中，所述载体***包括哺乳动物、细菌、酵母***等，并将包括质粒例如但不限于pALTER、pBAD、pcDNA、pCal、pL、 pELpGEMEX、pGEX、pCLpCMV、pEGFP、pEGFT，pSV2、pFUSE、pVITRO，pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2等其他可从实验室获得或市售的载体。适宜的载体可以包括质粒或病毒载体(例如，复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。
可以将包括编码所述抗体和其抗原结合片段的多核苷酸的载体引入宿主细胞用于克隆或基因表达。本发明中适用于克隆或表达所述载体中的DNA的宿主细胞为原核细胞、酵母或上述高级真核细胞。适用于本发明用途的原核细胞包括真细菌如，革兰氏阴性菌或革兰氏阳性菌，例如，肠杆菌科，如，大肠杆菌，肠杆菌属，欧文氏菌属，克雷白氏杆菌属，变形杆菌属，沙门氏菌属，如，鼠伤寒沙门(氏)杆菌，沙雷氏菌属，如，粘质沙雷氏菌，以及志贺氏菌属，及杆菌属如，枯草芽孢杆菌和地衣芽孢杆菌，假单胞菌如，绿肽杆菌和链霉菌。
除了原核细胞以外，真核微生物如丝状真菌或酵母也可作宿主细胞克隆或表达编码抗OX40抗体的载体。酿酒酵母，或面包酵母是最常用的低等真核宿主微生物。但是，许多其他属、种和株都比较常用且在本发明中适用，如粟酒裂殖酵母；克鲁维酵母属宿主如，乳酸克鲁维酵母、脆壁克鲁维酵母(ATCC12，424)、保加利亚克鲁维酵母(ATCC16，045)、魏氏克鲁维酵母(ATCC24，178)、克鲁雄酵母(ATCC56，500)、果蝇克鲁维酵母(ATCC36，906)、耐热克鲁维酵母和马克斯克鲁维酵母：解脂耶氏酵母(EP402，226)；巳斯德毕赤酵母(EP183，070)；假丝酵母：里氏木霉(EP244，234)；链孢霉；西方许旺酵母，如：西方许旺酵母；和丝状真菌，如：脉孢菌、青霉菌、弯颈霉和曲霉菌，如：钩巢曲霉和黑曲霉。
本发明中提供的适用于表达糖基化抗体或其抗原结合片段的宿主细胞由多细胞生物衍生得到。无脊椎细胞的实例包括植物和昆虫细胞。己发现多种杆状病毒株(baculoviral strains)及其变体以及对应的许可性昆虫宿主细胞(permissive insect host cells)，来自于诸如以下的宿主：草地夜蛾(毛虫)、埃及斑蚊(蚊子)、白纹伊蚊(蚊子)、黑腹果蝇(果蝇)及家蚕。多种用于转染的病毒株为公众可得，例如苜蓿银纹夜蛾核型多角体病毒和家蚕核型多角体病毒的Bm-5变种，这些病毒都可在本发明中使用，特别是用于转染草地夜蛾细胞。棉花、玉米、土豆、大豆、矮牵牛花、西红柿和烟草的植物细胞培养也可用作宿主。
但是，最感兴趣的是脊椎细胞，且脊椎细胞的培养(组织培养)己经成为常规操作。可用的哺乳动物宿主细胞实例有，SV40转化的猴肾细胞CV1系(COS-7，ATCC CRL 1651)；人胚胎肾细胞系(293或悬浮培养的293细胞亚 克隆，Graham et al.，].Gen Virol.36：59(1977))；幼地鼠肾细胞(B血，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.，Proc.Natl.Acad.Sci.USA 77：4216(1980))；小鼠睾丸支持细胞(TM4，Ma ther，Biol.Reprod.23：243-251(1980))；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人***细胞(HELA，ATCC CCL2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL75)；人肝细胞(Hep G2，HB 8065)；小鼠乳腺瘤(MMT 060562，ATCC CCL51)；TRI细胞(Ma ther等，Annals N.Y.Acad.Sci.383：44-68(1982))；MRC 5细胞；FS4细胞；及人肝癌细胞系(HepG2)。在某些优选的实施方式中，所述宿主细胞是293F细胞。
用上述的可产生抗OX40抗体的表达或克隆载体转化宿主细胞，并将其在常规的营养培养基中培养，所述营养培养基经修饰后适宜于诱导启动子、选择转化细胞或扩增编码目的序列的基因。
本发明中用于产生所述抗体或其抗原结合片段的宿主细胞可在多种本领域公知的培养基中培养。所述培养基还可含有本领域公知的适当浓度的任何其他必要的添加剂。所述培养基的条件，如温度、pH值等类似条件，为选择用于表达的宿主细胞此前所使用的条件，为普通技术人员所熟知。
在使用重组技术时，所述抗体可在胞内、壁膜空间生成，或直接分泌到培养基中。如果所述抗体在胞内生成，首先除去宿主细胞或裂解片断的颗粒残骸，例如，可通过离心或超声的方法。Carter et al.，Bio/Technology 10：163-167(1992)描述了将分泌到大肠杆菌壁膜空间的抗体分离的方法。简要地说，在醋酸铀(pH 3.5)、EDTA和苯甲磺酣氟(PMSF)存在的条件下化开细胞糊(cell paste)约30分钟以上。离心除去细胞碎片。如所述抗体分泌到培养基中，则通常首先使用市售的蛋白浓度过滤器，如lAmicon或Millipore Pellicon ultrafiltration unit，浓缩该表达***的上清液。在任何前述的步骤中都可加入蛋白酶抑制剂如PMSF以抑制蛋白降解，以及抗生素以防止偶然污染物的生长。
从所述细胞中制得的抗体可采用纯化方法进行纯化，例如羟磷灰石色谱、凝胶电泳、透析、DEAE-纤维素离子交换色谱柱、硫酸铵沉淀、盐析以及亲和色谱，其中亲和色谱为优选的纯化技术。所述抗体的种类以及所述抗体中存在任何免疫球蛋白的Fc结构域决定了蛋白A作为亲和配体是否适合。蛋白A可用于纯化基于人γ1，γ2或γ4重链的抗体(Lindmark et al.,J.lmmunol.Meth.62：卜13(1983))。蛋白G适用于所有鼠源异构体和人γ3(Guss et al.，EMBO J.5：1567 1575(1986))。琼脂糖是最常用的亲和配体附着基质，但也可选用其他基质。机械力稳定的基质如可控孔度玻璃或聚(苯乙 烯)苯与用琼脂糖相比可实现更快的流速和更短的处理时间。如该抗体含有CH3结构域，则可用Bakerbond ABX.TM树脂进行纯化(J.T.Baker，Phillipsburg，N.J.)。也可根据需要获得的抗体确定其他蛋白纯化的技术，如离子交换柱中的分馏、乙醇沉淀、反相HPLC、硅胶色谱、基于阴离子或阳离子交换树脂的肝素琼脂糖凝胶色谱(如聚天冬氨酸柱)、层析聚焦、SDS-PAGE、以及硫酸铵沉淀。
在任意初步纯化步骤之后，可用低pH疏水相互作用色谱的方法处理含有感兴趣的抗体和杂质的混合物，用pH约2.5-4.5的洗脱缓冲液，优选地在低盐浓度下进行(例如，从约0到0.25M盐浓度)。
试剂盒
本申请提供了包括所述抗OX40抗体和其抗原结合片段的试剂盒。在一些实施方式中，所述试剂盒用于检测在生物样品中的OX40的存在情况或水平。所述生物样品可以包括细胞或组织。
在一些实施方式中，所述试剂盒包括与可检测标记缀合的抗OX40抗体和其抗原结合片段。在一些实施方式中，所述试剂盒包括未标记的抗OX40抗体和其抗原结合片段，并进一步包括能够与未标记的抗OX40抗体和其抗原结合片段结合标记的二抗。所述试剂盒可以进一步包括使用说明和在试剂盒中将每个组件分隔开的包装。
在一些实施方式中，所述抗OX40抗体和其抗原结合片段与底物或仪器连接用于夹心测定如ELISA或免疫色谱测定。适用的底物或仪器可以是例如微孔板和试纸。
药物组合物和治疗方法
本申请进一步提供了包括所述抗OX40抗体和其抗原结合片段的药物组合物和一个或多个药学上可接受的载体。
用在本申请公开的药物组合物中的药用可接受载剂可包括，例如，药用可接受的液体、凝胶或固体载剂、水相介质、非水相介质、抗微生物物质、等渗物质、缓冲液、抗氧剂、麻醉剂、悬浮剂/分散剂、整合剂、稀释剂、佐剂、辅料或无毒辅助物质，其他本领域公知的组分或以上的多种组合。
适用的组分可包括，例如，抗氧剂、填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、矫味剂、增稠剂、着色剂、乳化剂或稳定剂例如糖和环糊精。适用的抗氧剂可包括，例如，甲硫氨酸、抗坏血酸、EDTA、硫代硫酸纳、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙酸、巯基山梨醇、丁基甲基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。如本发明所公开，在一种含有本发明公开的抗体或其抗原结合片段的组合物中包括一种或多 种抗氧剂如甲硫氨酸，可将降低所述抗体或其抗原结合片段的氧化。对氧化作用的减少可防止或减少结合亲和力的降低，从而提高抗体稳定性并延长保质期。因此，在某些实施方式中，本发明提供的组合物中含有一种或多种所述的抗体或其抗原结合片段以及一种或多种抗氧剂例如甲硫氨酸。本发明进一步提供了多种方法，通过将本发明中提供的抗体或其抗原结合片段与一种或多种抗氧剂混合，例如甲硫氨酸，可防止所述抗体或其抗原结合片段氧化、延长其保质期和/或提高其活性。
进一步的说，药用可接受的载剂可包括，例如，水相介质如氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、或葡萄糖和乳酸林格注射液、非水介质例如：植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油、细菌抑制或真菌抑制浓度下的抗菌物质、等渗剂如：氯化钠或葡萄糖、缓冲液如：磷酸盐或枸橼酸盐缓冲液，抗氧化剂如：硫酸氢钠，局部麻醉剂如：盐酸普鲁卡因，助悬剂和分散剂如：羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如：聚山梨醇酯80(吐温-80)、整合试剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2一氨基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。作为载剂的抗菌剂可加入多次剂量容器中的药物组合物中，其包括酚类或甲酚、汞制剂、苯甲醇、氯代丁醇、甲基和丙基对羟基苯甲酸酯、噻汞撒、氯苯甲烷铵和氯苯乙铵。适用的辅料可包括，例如，水、盐、葡萄糖、甘油或乙醇。适用的无毒辅助物质可包括，例如，乳化剂、pH值缓冲剂、稳定剂、增溶剂，或者醋酸钠、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或者环糊精之类的物质。
所述药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊、片剂、持续释放制剂或粉末。口服制剂可以包括标准载体如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯皖酮、糖精钠、纤维素、碳酸镁等。
在某些实施方式中，所述药物组合物被制剂成可注射的组合物。可注射的药物组合物可以任何常规的形式制备，例如，液体溶剂、悬浮剂、乳化剂或适用于产生液体溶剂、悬浮剂或乳化剂的固体形式。注射制剂可包括现用的无菌和/或无热原溶液、使用前现与溶剂结合的无菌干燥的可溶物，如冻干粉，包括皮下片、注射即用的无菌悬浮剂、使用前现与介质结合的无菌干燥不溶产品，和无菌和/或无热原的乳剂。溶剂可以为水相或非水相。
在某些实施方式中，单位剂量的注射制剂包装在一个安瓿、一支管或一支带有针的针筒中。本领域悉知，所有注射给药的制剂应为无菌无热原。
在某些实施方式中，通过将本申请公开的抗体或其抗原结合片段溶解于某适当的溶剂中可制备无菌冻干的粉末。所述溶剂可含有一种可提高粉或由 粉末制得的重组溶液的稳定性，或改善粉末或重组溶液的其他药理组分。适用的辅料包括，但不限于，水、葡萄糖、三梨糖醇、果糖、玉米糖浆、木糖醇、甘油、葡萄糖、黑糖或其他适用的物质。溶剂可含有缓冲液，如枸橼酸缓冲液、磷酸钠或磷酸钾缓冲液或其他本技术熟练人员公知的缓冲液，在一种实施方式中，缓冲液的pH为中性。在本领域公知的标准条件下进行对所述溶液进行随后的过滤除菌，然后冻干制得理想的制剂。在一种实施方式中，将所得的溶剂分装至小管中冻干。每支小管可容纳单次剂量或多次剂量的所述抗OX40抗体或其抗原结合片段或其组合物。每支小管中的装入量可略微高于每次剂量所需或多次剂量所需(例如10％过量)，从而保证取样精确和给药精确。冻干粉可在适当的条件下储存，如在约4℃到室温范围。
用注射用水将冻干粉重溶得到用于注射给药的制剂。在一种实施方式中，可将冻干粉加至无菌无热原水或其他适用的液体载剂中重溶。精确的量由选择的疗法决定，可根据经验值决定。
还提供了治疗方法，包括将治疗有效量的本申请所述的抗OX40抗体或其抗原结合片段施用给需要其的受试者，由此治疗或预防与OX40相关的状况或病症。在另一方面，还提供了治疗会从上调的免疫响应获益的受试者的状况的方法，包括对所述需要其的受试者施用治疗有效量的本申请所述的抗OX40抗体或其抗原结合片段。
本申请中提供的抗OX40抗体或其抗原结合片段的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、现用治疗、对象的健康状况和交叉感染的潜力、过敏、超敏和副作用，以及给药途径和肿瘤发展的程度。本领域熟练人员(例如医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。
在某些实施方式中，本发明提供的抗OX40抗体或其抗原结合片段可在治疗有效剂量约0.01mg/kg到约100mg/kg之间给药。在某些实施方式中，所述抗OX40抗体或其抗原结合片段以约50mg/kg或更少的剂量给药，在某些实施方式中，给药剂量为10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少或0.1mg/kg或更少。某特定剂量可在多个间隔给药，例如每天一次、每天两次或更多、每月两次或更多、每周一次、每两周一次、每三周一次、每月一次或每两月或更多月一次。在某些实施方式中，给药剂量可随治疗进程变化。例如，在某些实施方式中，初始给药剂量可比后续给药剂量高。在某些实施方式中，给药剂量在治疗进程中根据给药对象的反应进行调整。
给药方案可通过调整达到最优反应(如治疗反应)。例如，可进行单剂量给药或在二段时间分多个分隔的剂量给药。
本发明中公开的抗体和抗原结合片段可通过本领域公知的给药方式给药，例如注射给药(如，皮下注射、腹腔注射、静脉注射，包括静脉滴注，肌肉注射或皮内注射)或非注射给药(如，口服给药、鼻腔给药、舌下给药、直肠给药或外用给药)。
与OX40相关的状况和病症可以是免疫相关的疾病或病症。在某些实施方式中，所述与OX40相关的状况和病症包括肿瘤和癌症、例如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、***癌、成胶质细胞瘤、子***、胸腺癌、白血病、淋巴瘤、骨髓瘤、草样肉芽肿(mycoses fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经***(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。在某些实施方式中，所述肿瘤和癌症是转移性的，尤其是表达OX40的转移性肿瘤。在某些实施方式中，所述与OX40相关的状况和病症包括慢性病毒感染，例如乙型肝炎，丙型肝炎，疱疹病毒，Epstein-Barr病毒，艾滋病毒，巨细胞病毒，单纯疱疹病毒I型，单纯疱疹病毒2型，人***状瘤病毒，腺病毒的病毒感染，卡波西肉瘤相关的疱疹病毒流行病，薄环病毒(Torquetenovirus)，JC病毒或BK病毒等。
使用方法
本申请进一步提供了使用所述抗OX40抗体或其抗原结合片段的方法。
在一些实施方式中，本申请提供了在个体中治疗与OX40相关的状况或病症的方法，包括施用治疗有效量的本申请所述的抗OX40抗体或其抗原结合片段。在一些实施方式中，所述个体被鉴定为患有可能对OX40激动剂响应的病症或状况。
在目标生物组织中OX40的存在情况和水平可以指示所述生物样品来源的个体是否可能对OX40激动剂响应。可以使用多种方法在来自所述个体的待测生物样品中确定OX40的存在情况或水平。例如，可以将所述待测生物样品暴露于抗OX40抗体或其抗原结合片段，其与表达的OX40蛋白结合并检测表达的OX40蛋白。在一些实施方式中，所述待测样品来源于癌细胞或组织，或进入肿瘤的免疫细胞。在一些实施方式中，在所述待测生物样品中OX40的存在或水平上调表示响应的可能性。本申请使用的术语"上调"是指 与使用相同抗体检测的参照样品中OX40蛋白水平相比，使用本申请所述的抗体或其抗原结合片段在待测样品中检测的OX40蛋白水平的总的增加不少于10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％或更多。所述参照样品可以是从健康或无疾病的个体中获得的对照样品，或从待测样品来源的个体中获得的健康或无疾病的样品。例如，所述参照样品可以是在待测样品(如肿瘤)附近或相邻的无疾病样品。
本发明公开的抗体和抗原结合片段可单独给药或与一种或多种其他治疗手段或物质联合给药。例如，本发明公开的抗体和抗原结合片段可与化疗、放疗、癌症治疗手术(如肿瘤切除术)、一种或多种抗呕吐药或其他化疗导致的并发症的疗法、或任何其他用于癌症的治疗物质或任何由OX40介导的病症的治疗物质进行联用。在某些这样的实施方式中，本发明公开的抗体和抗原结合片段与一种或多种治疗物质联用时，可与所述的一种或多种治疗物质同时给药，在某些这样的实施方式中，所述的抗体和抗原结合片段可作为同一个药物组合物的一部分同时给药。但是，与其他治疗物质"联用"的抗体和抗原结合物不需要同时给药或与该治疗物质在同一组合物中给药。本发明中"联用"的含义还包括在另一个治疗物质之前或之后给药的抗体和抗原结合物也被认为是与该治疗物质"联用"，即使所述抗体或其抗原结合片段与第二种物质通过不同给药方式给药。在可能的情况下，与本发明公开的抗体或其抗原结合片段联用的其他治疗物质可参照该其他治疗物质的产品说明书的方法用药，或参照、外科医生的案头参考书2003(Physicians'Desk Reference，57th Ed；Medical Economics Company；ISBN：1563634457；第57版(2002年11月))，或参照其他本领域公知的方法。
在某些实施方式中，所述治疗物质能够诱导或增强针对癌症的免疫反应。例如，肿瘤疫苗可以用于诱导对某些肿瘤或癌症的免疫应答。细胞因子治疗可以用于提高将肿瘤抗原向免疫***的递呈。细胞因子治疗的示例包括但不限于干扰素如干扰素α、β和γ，集落剌激因子如巨噬细胞CSF、粒细胞巨噬细胞CSF和粒细胞CSF，白介素如1L-L、1L-1a、1L-2、1L-3、1L-4、1L-5、1L-6、1L-7，1L-8、1L-9、1L-10、1L-ll和1L-12，肿瘤坏死因子如TNF-α和TNF-β。还可以使用灭活免疫抑制目标的试剂，如PD-L1/PD-1抗体、TGF-β抑制剂、IL-10抑制剂和Fas配体抑制剂。另一组试剂包括激活针对肿瘤或癌细胞的免疫响应的那些试剂，例如，提高T细胞激活(如T细胞共剌激分子激动剂如CTLA-4、ICOS)的那些，以及提高树突细胞功能和抗原递呈的那些。
本申请进一步提供了在用OX40激动剂治疗的受试者中监测治疗反应或 疾病进展的方法，包括用本申请所述抗OX40抗体或其抗原结合片段在来自所述个体的待测生物样品中确定OX40的存在情况或水平。在某些实施方式中，所述方法进一步包括将待测生物样品中的OX40水平与此前从同一个体上获得的可比样品中的OX40水平进行比较，其中在测试生物样品中OX40水平的增加减少或减慢或停止，表明积极的治疗反应或受控的疾病进展。所述可比样品可以与待测样品是同一类型的样品，但其是在治疗前或在治疗初期阶段从相同个体中获得的。
以下实施例旨在更好地说明本发明，且不应理解为限制本发明的范围。所有下述的特定组合物、材料和方法，其整体或部分，都在本发明的范围内。这些特定的组合物、材料和方法不是为了限制本发明，而只是为说明特定的实施方式在本发明的范围内。本领域熟练技术人员可不添加创造性及不偏离本发明范围而开发出等同的组合物、材料和方法。应理解，在对本发明的方法作出的多种改动可以仍然包括在本发明范围内。发明人意在将这样的变动包括在本发明的范围内。
实施例1：抗人OX40激活性单克隆抗体的获得
本申请发明人构建了过表达OX40的CHO细胞株，以之免疫小鼠，并进行杂交瘤细胞融合。通过抗原抗体结合实验和细胞功能筛选，得到了一系列具有较强ELISA结合活性和OX40激活功能的鼠源抗体。经测序获得各抗体的可变区VH和VL的序列，并据此设计和表达了人-鼠嵌合抗体，其所述重链互补决定区选自以下序列：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11中的一个或多个。所述轻链互补决定区选自以下序列：SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14中的一个或多个。
更为优选地，所述重链互补决定区为：SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；或SEQ ID NO：9、SEQ ID NO：10和/或SEQ ID NO：11。所述轻链互补决定区为：SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：6；SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8；或SEQ ID NO：12、SEQ ID NO：13和/或SEQ ID NO：14。
本发明较优实施例中，重链互补决定区及轻链互补决定区分别如表1所示，
a.重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3；轻链互补决定区包括：SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
b.重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO： 3；轻链互补决定区包括：SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
c.重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3；轻链互补决定区包括：SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；
d.重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3；轻链互补决定区包括：SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；或重链互补决定区包括SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11；轻链互补决定区包括：SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14。
具体地，本实施例1中发明人构建了过表达OX40的CHO细胞株，以之免疫小鼠，并与SP2/0-AG14细胞株进行杂交瘤细胞融合。通过抗原抗体结合实验和细胞功能筛选，得到了一系列具有较强ELISA结合活性和OX40激活功能的鼠源抗体。经测序获得各抗体的可变区VH和VL的序列，并据此设计和表达了人-鼠嵌合抗体，包括MT01-L1和MT01-L2。
为增强抗体稳定性，减少可能的氧化及异构化对抗体纯度和活性的影响，对抗体重链和轻链V区的潜在的不稳特定位点进行了突变。例如，将MT01-L1的重链第57氨基酸Glycin突变为Alanine，将MT01-L1的轻链第96位氨基酸Tryptophan突变为phenylalanine。所得突变抗体为MT01-L1(M1)，MT01-L1(M2)，MT01-L1(M1/M2)。
为了消除抗体的ADCC、CDC活性，将抗体重链的亚型由人IgG1置换为人IgG2亚型，所得嵌合抗体为MT01-L1(G2)
将MT01-L1和MT01-L1(G2)的重链和轻链可变区进行人源化后，所得人源化抗体为MT01-C1和MT01-C1(G2)。
实施例2：抗体的制备
将编码融合蛋白的重链和轻链的cDNA序列分别克隆到哺乳动物细胞表达载体pcDNA3.4上。将重链表达质粒和轻链表达质粒按2：1的摩尔比用Lipofectamine 2000转染试剂(Invitrogen)转染入HEK293细胞，并在37℃、5％二氧化碳条件下培养7天。收集培养液上清，并用Protein A亲和层析法提纯上清中的抗体。纯化后的抗体经PBS溶液透析和冷冻干燥浓缩后，保存于-20℃。
实施例3：ELISA结合实验
将浓度为1μg/mL的蛋白溶液以100μL/well包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/well的5％BSA/PBS封闭2小时，室温振荡。300μL PBST洗涤3次。用PBS配制样品的梯度稀释溶液。以100μL/well加入96孔板，室温振荡1小时。300μL PBST洗涤3次。配制二抗goat anti-human IgG HRP溶液，以 100μL/well加入96孔板，室温振荡1小时。300μLPBST洗涤4次。加入100μL/well TMB，显色20min。加入100μL/well 0.6N H2SO4，终止显色，检测OD450nm。
经检测，结果如图1所示，人-鼠嵌合抗体MT01-L1和MT01-L2的ELISA结合EC50分别为290.7ng/mL和208.5ng/mL。
实施例4：与CHO-hOX40的结合
用PBS配置OX40抗体梯度，配制成终浓度的10×工作液。收集CHO-hOX40细胞，PBS洗涤一遍后计数，稀释成2*10^6/ml细胞悬液；分别取10μlOX40抗体工作液加入100μl细胞悬液中，4℃避光孵育30min；PBS洗涤2次后，加入二抗，4℃避光孵育30min，PBS洗涤一次后，以400μl FACS buffer悬起，上机检测。如图2所示，结果显示MT01-L1和MT01-L2与人OX40有结合，其EC50分别为1.22μg/mL和2.42μg/mL。
类似的，本申请发明人检测了抗体与表达了小鼠OX40的CHO细胞的结合，参见图3的结合实验结果，发现MT01-L1和MT01-L2与小鼠OX40都没有结合。
实施例5：OX40信号通路激活实验
本申请发明人构建了检测OX40激活性抗体功能的细胞实验体系。具体来说，本申请发明人构建了Jurkat-OX40-NFκB-luciferase reporter稳转细胞株，当把OX40激活性抗体与该稳转细胞株及过表达FcR的HEK293细胞混合后，OX40抗体可以激活NFκB-luciferase报告基因的表达。
用PBS配置OX40抗体梯度，配制成终浓度的2×工作液，冰上操作。收集Jurkat-NFkB-luc-OX40细胞和过表达FcR的HEK293细胞，离心后重悬于培养基中。于384孔板中，加入OX40Ab和适量细胞悬液，孵育5小时后，加入One-Glo(Promega)检测试剂，混匀后用Pherastar检测荧光信号。
如图4(a)所示，经检测，MT01-L1、MT01-L1(G2)、MT01-L2、MT01-L2(G2)在上述实验体系中激活NFκB-luciferase报告基因的EC50分别为39.1、14.9、84.1和117.9ng/mL。
如图4(b)所示，经检测，MT01-L1、MT01-L1(M1)、MT01-L1(M2)、MT01-L1(M1/M2)在上述实验体系中激活NFκB-luciferase报告基因的EC50分别为57.35、64.52、59.95和127.9ng/mL。
实施例6：与OX-40L竞争实验
用PBS稀释OX40L，配制成终浓度的10×工作液，用PBS配置OX40纯化抗体梯度，配制成终浓度的2×工作液，冰上操作。收集Jurkat-NFkB-luc-OX40细胞，离心后重悬于培养基中，配成细胞悬液。于384孔板中，加入OX40Ab、细胞悬液和OX40L。37℃，5％CO2培养箱内孵育 5小时后，加入One-Glo(Promega)试剂，混匀后用Pherastar检测荧光信号。如图5所述，结果显示MT01-L1与MT01-L2对OX40L诱导的Jurkat细胞NFkB信号通路活化的抑制IC50分别为0.686μg/mL和1.59μg/mL。
实施例7：ADCC实验
将CHO-OX40-luc铺于96孔平底板上，培养过夜；第二天用1640培养基配制OX40Ab梯度稀释液，并加入到96孔平底板内。加入人PBMC共孵育48h后，离心取上清，用Cyto-Tox Glo试剂盒检测。如图6所示，结果显示嵌合抗体MT01-L1和MT01-L2在大于1μg/mL浓度时都有明显的ADCC作用。
实施例8：OX40抗体促进原代Th细胞增殖
从健康志愿者血液中分离CD4+细胞，用含PHA/IL-2的1640完全培养基培养两天待用。将OKT3(0.1μg/孔)和经过梯度稀释的OX40抗体或是对照IgG包被于高亲和力96孔圆底板中。第二天，用PBS将孔板洗涤2次，将经过预处理的CD4+细胞按50000每孔加入到预包被了抗体的96孔板中，继续培养7天后用CCK8试剂盒检测细胞增殖情况。如图7所示，实验结果显示，OKT3抗体存在的情况下，OX40抗体可以剂量依赖的促进CD4 Th细胞的增殖。实验中同时检测了只加了最高浓度1500ng/mL的OX40抗体的情况下对CD4+细胞的活力，并以此为基础标准化处理了数据。
实施例9：嵌合抗体动物药效实验
按10 6细胞/只的量将鼠结肠癌细胞接种于OX40人源化小鼠(OX40胞外段置换为人OX40胞外段序列)皮下。待肿瘤长至约100mm 3后，静脉给予OX40抗体或IgG1对照(10mg/kg)，每3天给药一次，共给药4次。如图8(a)和图8(b)结果显示，在开始给药13天后，与对照组相比MT01-L1具有极显著药效。而在相同给药方案下，MT01-L2的药效虽然没有达到统计学显著性差异，但也显示出明显的药效趋势。
实施例10：人源化抗体活性
通过对嵌合抗体做CDR grafting和氨基酸回复突变，获得了人源化抗体MT01-C1和MT01-C1(G2)。
人源化抗体与人OX抗原蛋白的体外结合实验如实施例3中描述的方法进行。结果显示人源化抗体与人OX40蛋白的ELISA结合EC50分别为0.345μg/mL和0.390μg/mL
人源化抗体体外活性实验如实施例5中描述的方法进行。结果显示MT01-C1和MT01-C1(G2)在Jurkat-OX40-NFκB-luciferase reporter稳转细胞株上的激活EC50分别为0.028和0.0434μg/mL(图9b)。
人源化抗体的动物药效实验如实施例8中的方法进行，但是抗体给药方式改为15mg/kg，单次给药。结果显示给药17天后，MT01-C1和MT01-C1(G2)对肿瘤体积的抑制率分别达到了79％和66％(图9c)
实施例11：人源化抗体小鼠及食蟹猴PK性质
在小鼠PK实验中，每个抗体按5mg/kg剂量静脉注射C56BL6小鼠，雌性，6只/组。给药后1h，2h，6h，24h，48h，72h，96h，192h，312h，每个时间点每组采3只小鼠100μL血样，4度静置2-3h后，5000rpm离心10min，取血清，-80℃保存，用于ELISA检测。
为检测血清中抗体浓度，将高亲和力微孔板包被人OX40抗原。小鼠血清样品经稀释缓冲液1：100稀释后加入孔内，C57BL6小鼠血清中的MT01-C1和MT01-C1(G2)与微孔板中包被的人OX40-6his结合，未结合的药物在洗板步骤中被洗去。再加入标记了辣根过氧化物酶(HRP)的驴抗人IgG，其与抗原抗体复合物结合，经过洗涤后再加入HRP酶的底物(TMB)，产生颜色反应，颜色的深浅和待测抗体的浓度呈正比。1M H2SO4终止反应后用酶标仪读取OD值，检测波长450nm，参比波长620nm。通过对比OD450nm读值与样品标准曲线计算生物样品中待测抗体的浓度。
检测结果显示MT01-C1与MT01-C1(G2)的Cmax分别为125.7μg/mL和107.2μg/mL，t 1/2分别为265hs和321hs。(图10a)
在食蟹猴PK实验中，每个抗体按1.5mg/kg剂量静脉注射雄性食蟹猴，一组3只。动物组给药前48h，给药后15min，30min，1，2，6，24，48，72，96，168，240，336，504h，每个时间点各采100ul血样，4度静置2-3h后，5000rpm离心10min，取血清，-80℃保存，用于ELISA检测。
为检测血清中抗体浓度，将高亲和力微孔板包被人OX40抗原。食蟹猴血清样品经稀释缓冲液1：100稀释后加入孔内，食蟹猴血清中的MT01-C1和MT01-C1(G2)与微孔板中包被的人OX40-6his结合，未结合的药物在洗板步骤中被洗去。再加入标记了辣根过氧化物酶(HRP)的驴抗人IgG，其与抗原抗体复合物结合，经过洗涤后再加入HRP酶的底物(TMB)，产生颜色反应，颜色的深浅和待测抗体的浓度呈正比。1M H2SO4终止反应后用酶标仪读取OD值，检测波长450nm，参比波长620nm。通过对比OD450nm读值与样品标准曲线计算生物样品中待测抗体的浓度。
检测结果显示食蟹猴静脉注射1.5mg/kg后，末端消除项t1/2为 121.307±66.853hr，Cmax为42.619±3.464μg/mL。(图10b)
上述仅为本发明的优选实施例而已，并不对本发明起到任何限制作用。任何所属技术领域的技术人员，在不脱离本发明的技术方案的范围内，对本发明揭露的技术方案和技术内容做任何形式的等同替换或修改等变动，均属未脱离本发明的技术方案的内容，仍属于本发明的保护范围之内。

Claims (23)

  1. 一种分离的抗体或其抗原结合片段，其特征在于，所述分离的抗体或其抗原结合片段包括重链互补决定区和轻链互补决定区，所述重链互补决定区选自以下序列：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11中的一个或多个。
  2. 根据权利要求1所述的分离的抗体或其抗原结合片段，其特征在于，所述重链互补决定区为：SEQ ID NO：1、SEQ ID NO：2和/或SEQ ID NO：3；
    SEQ ID NO：1、SEQ ID NO：7和/或SEQ ID NO：3；或
    SEQ ID NO：9、SEQ ID NO：10和/或SEQ ID NO：11。
  3. 根据权利要求1或2所述的分离的抗体或其抗原结合片段，其特征在于，所述轻链互补决定区选自以下序列：SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14中的一个或多个。
  4. 根据权利要求3所述的分离的抗体或其抗原结合片段，其特征在于，所述轻链互补决定区为：
    SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：6；
    SEQ ID NO：4、SEQ ID NO：5和/或SEQ ID NO：8；或
    SEQ ID NO：12、SEQ ID NO：13和/或SEQ ID NO：14。
  5. 根据权利要求1-4之任一项所述的分离的抗体或其抗原结合片段，其特征在于，
    重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3；轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
    重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3；轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
    重链互补决定区包括SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3；轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；
    重链互补决定区包括SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3； 轻链互补决定区包括SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：8；或
    重链互补决定区包括SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11；轻链互补决定区包括SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14。
  6. 根据权利要求1-5之任一项所述的分离的抗体或其抗原结合片段，其特征在于，所述重链可变区替换为以下序列：SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：29。
  7. 根据权利要求6所述的分离的抗体或其抗原结合片段，其特征在于，所述轻链可变区替换为以下序列：SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：30。
  8. 根据权利要求6-7所述的分离的抗体或其抗原结合片段，其特征在于，
    重链可变区包括SEQ ID NO：15；轻链可变区包括SEQ ID NO：16；
    重链可变区包括SEQ ID NO：17；轻链可变区包括SEQ ID NO：16；
    重链可变区包括SEQ ID NO：15；轻链可变区包括SEQ ID NO：18；
    重链可变区包括SEQ ID NO：17；轻链可变区包括SEQ ID NO：18；或
    重链可变区包括SEQ ID NO：19；轻链可变区包括SEQ ID NO：20。
  9. 根据权利要求1-8之任一项所述的抗体或其抗原结合片段，其是人源化或是全人源单克隆抗体。
  10. 根据权利要求1-8之任一项所述的抗体或其抗原结合片段，其是骆驼化单域抗体、双功能抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体、纳米抗体、域抗体或双价域抗体。
  11. 根据权利要求1-8之任一项所述的抗体或其抗原结合片段，其进一步包括免疫球蛋白恒定区，包括人IgG1、IgG2或IgG4的蛋白的恒定区。
  12. 根据权利要求1-8之任一项所述的抗体或其抗原结合片段，其进一步包括缀合物。
  13. 一种分离的多核苷酸，其编码根据权利要求1-8中任一项所述的抗体或其抗原结合片段。
  14. 一种载体，其包括根据权利要求13所述的分离的多核苷酸。
  15. 一种宿主细胞，其包括根据权利要求14所述的载体。
  16. 一种表达根据权利要求1-12中任一项所述的抗体或其抗原结合片段的方法，其包括在表达根据权利要求13所述的分离的多核苷酸的条件下培养根据权利要求15所述的宿主细胞。
  17. 一种试剂盒，其包括根据权利要求1-12中任一项所述的抗体或其抗原结合片段。
  18. 一种药物组合物，包括根据权利要求1-12中任一项所述的抗体或其抗原结合片段以及一种或多种药学上可接受的载体。
  19. 权利要求1-12之任一项所述的抗体或其抗原结合片段在检测人或猴OX40的存在或水平中的应用。
  20. 权利要求1-12之任一项所述的抗体或其抗原结合片段在检测鉴别患有对OX40激动剂响应的病症或状况的个体中的应用。
  21. 权利要求1-12之任一项所述的抗体或其抗原结合片段在监测OX40激动剂治疗的治疗反应或疾病进展中的应用。
  22. 根据权利要求1-12中任一项所述的抗体或其抗原结合片段在制备用于治疗会从上调的免疫响应中获益的状况的药物中的用途。
  23. 根据权利要求22所述的用途，其中所述状况是癌症或慢性病毒感染。
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VALZASINA BGUIDUCCI CDISLICH HKILLEEN NWEINBERG ADCOLOMBO MP.: "Triggering of OX40 (CD134) on CD4(+)CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR", BLOOD, vol. 105, 2005, pages 2845 - 51, XP055216233, DOI: 10.1182/blood-2004-07-
VETTO JTLUM SMORRIS ASICOTTE MDAVIS JLEMON MWEINBERG A.: "Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers", AM J SURG., vol. 174, 1997, pages 258 - 65, XP002109414, DOI: 10.1016/S0002-9610(97)00139-6
VOO KSBOVER LHARLINE MLVIEN LTFACCHINETTI VARIMA KKWAK LWLIU YJ.: "Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function", J IMMUNOL., vol. 191, 2013, pages 3641 - 50, XP055194259, DOI: 10.4049/jimmunol.1202752
VU MDXIAO XGAO WDEGAUQUE NCHEN MKROEMER AKILLEEN NISHII NLI XC.: "OX40 costimulation turns off Foxp3+ Tregs", BLOOD, vol. 110, 2007, pages 2501 - 10, XP055443260, DOI: 10.1182/blood-2007-01-070748
WEINBERG ADRIVERA MMPRELL RMORRIS ARAMSTAD TVETTO JTURBA WJALVORD GBUNCE CSHIELDS J.: "Engagement of the OX-40 receptor in vivo enhances antitumor immunity", J IMMUNOL., vol. 164, 2000, pages 2160 - 9, XP002224132
WILLOUGHBY JGRIFFITHS JTEWS ICRAGG MS.: "OX40: Structure and function-What questions remain?", MOL IMMUNOL., vol. 83, 2017, pages 13 - 22, XP029925307, DOI: 10.1016/j.molimm.2017.01.006
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ZHANG LCONEJO-GARCIA JRKATSAROS DGIMOTTY PAMASSOBRIO MREGNANI GMAKRIGIANNAKIS AGRAY HSCHLIENGER KLIEBMAN MN: "Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer", N ENGL J MED., vol. 348, 2003, pages 203 - 13, XP002390426, DOI: 10.1056/NEJMoa020177

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US11440965B2 (en) 2022-09-13
EP3733703A1 (en) 2020-11-04
AU2018393315A1 (en) 2020-07-30
AU2018393315A2 (en) 2021-02-25
CN108218990A (zh) 2018-06-29
KR20200106052A (ko) 2020-09-10
JP2021514198A (ja) 2021-06-10
US20210269539A1 (en) 2021-09-02
JP7317858B2 (ja) 2023-07-31
CN108218990B (zh) 2021-03-02
EP3733703A4 (en) 2021-12-29

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