WO2020119793A1 - Anticorps humanisés dirigés contre ox40, procédé de préparation correspondant, et utilisation associée - Google Patents

Anticorps humanisés dirigés contre ox40, procédé de préparation correspondant, et utilisation associée Download PDF

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WO2020119793A1
WO2020119793A1 PCT/CN2019/125251 CN2019125251W WO2020119793A1 WO 2020119793 A1 WO2020119793 A1 WO 2020119793A1 CN 2019125251 W CN2019125251 W CN 2019125251W WO 2020119793 A1 WO2020119793 A1 WO 2020119793A1
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seq
antibody
antigen
binding portion
set forth
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PCT/CN2019/125251
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Yong Zheng
Baotian YANG
Jing Li
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Wuxi Biologics (Shanghai) Co., Ltd.
WuXi Biologics Ireland Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application generally relates to antibodies. More specifically, the application relates to humanized monoclonal antibodies against OX40, a method for preparing the same, and the use thereof.
  • Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as OX40, CD134 and ACT35) , one of the immune-checkpoint proteins, plays a major role in T cell function by potentiating T cell receptor signaling and leading to their activation.
  • OX40 is primarily expressed by activated CD4+ and CD8+ T cells, memory T cells, regulatory T (Treg) cells and nature killer (NK) cells.
  • the interaction of OX40 expressed on activated T cells, and its ligand (OX40L) expressed on antigen presenting cells dramatically promotes T cell activation, proliferation and migration, increases survival of effector T cells, enhances the germinal center formation and dendritic cells maturation.
  • OX40 signaling can inhibit differentiation and expansion of Tregs, antagonize generation of inducible Tregs and block Treg-suppressive function. It has been proved in a variety of preclinical mouse tumor models and clinical trials that agonist of OX40 is quite a promising strategy for treating cancer and infectious diseases.
  • agonistic agents targeting OX40 have been developed by big pharmaceutical companies, such as MedImmune, GlaxoSmithKline (GSK) , Pfizer and Incyte.
  • An agonistic murine antibody targeting OX40 (9B12, AgonOX) was used in Phase I clinical trial in patients with advanced cancer. Patients treated with one course of the mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell response to reporter antigen immunizations (e.g.
  • GSK is also developing GSK-3174998, a humanized IgG1 monoclonal antibody that activates OX-40 on the surface of T cells, identified through a collaboration with MD Anderson Cancer Center, for the potential treatment of cancer including solid tumors and hematological malignancies.
  • target OX40 include Pfizer’s fully human IgG2 agonist antibody PF-04518600, which is currently in clinical development in a broad spectrum of malignancies; and Incyte’s INCAGN-1949, which is an anti-OX40 human IgG1 antibody with optimal agonistic profile and the ability of selectively deplete intratumoral regulatory T cells, for the potential treatment of cancer.
  • humanized antibodies against OX40 haven been generated utilizing proprietary hybridoma technology.
  • the antibodies of the present disclosure have high binding affinity; specifically bind to both human and monkey OX40 protein without cross-family reactions; and potent modulating immune responses, including enhancing T cell proliferation and increasing cytokine IFN ⁇ and interleukin-2 production.
  • the present disclosure provides humanized monoclonal antibodies against OX40. It also provides the methods of hybridoma generation using Balb/c mice, the nucleic acid molecules encoding the anti-OX40 antibodies, expression vectors and host cells used for the expression of anti-OX40 antibodies. The present disclosure further provides the methods for validating the function of antibodies in vitro.
  • the antibodies of the present disclosure provide a very potent agent for the treatment of multiple cancers vial modulating human immune function.
  • the present disclosure comprises an isolated antibody, or an antigen-binding portion thereof.
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • HCDRs heavy chain CDRs
  • HCDR2 comprising one of the sequences selected from the group consisting of SEQ ID NOs: 3, 7 and 8;
  • LCDRs light chain CDRs
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • HCDRs heavy chain CDRs
  • HCDR2 as set forth in one of the sequences selected from the group consisting of SEQ ID NOs: 3, 7 and 8;
  • LCDRs light chain CDRs
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • an isolated antibody or the antigen-binding portion thereof of the present disclosure competes binding for the same epitope with the isolated antibody or the antigen-binding portion thereof as defined above.
  • the present disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region and/or the light chain variable region of the isolated antibody as disclosed herein.
  • the present disclosure is directed to a vector comprising the nucleic acid molecule encoding the antibody or antigen -binding portion thereof as disclosed herein.
  • the present disclosure is directed to a host cell comprising the expression vector as disclosed herein.
  • the present disclosure is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.
  • the present disclosure is directed to a method for preparing an anti-OX40 antibody or antigen-binding portion thereof which comprises expressing the antibody or antigen-binding portion thereof in the host cell and isolating the antibody or antigen-binding portion thereof from the host cell.
  • the present disclosure is directed to a method of modulating an immune response in a subject, comprising administering the antibody or antigen-binding portion thereof as disclosed herein to the subject such that an immune response in the subject is modulated.
  • the present disclosure is directed to a method for treating abnormal cell growth in a subject, administering an effective amount of the antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.
  • the present disclosure is directed to a method for inhibiting growth of tumor cells in a subject, administering an effective amount of the antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.
  • the present disclosure is directed to a method for reducing tumor cell metastasis in a subject, administering an effective amount of the antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.
  • the present disclosure is directed to a method for treating or preventing diseases comprising proliferative disorders (such as cancers) , autoimmune diseases, inflammatory disease or infectious diseases in a subject comprising administering an effective amount of the antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.
  • the present disclosure is directed to the use of the antibody or antigen-binding portion thereof as disclosed herein in the manufacture of a medicament for treating or preventing diseases comprising proliferative disorders (such as cancers) , autoimmune diseases, inflammatory disease or infectious diseases.
  • the present disclosure is directed to the use of the antibody or antigen-binding portion thereof as disclosed herein in the manufacture of a diagnostic agent for diagnosing proliferative diseases comprising proliferative disorders (such as cancers) , autoimmune diseases, inflammatory disease or infectious diseases.
  • the present disclosure is directed to the antibody or antigen-binding portion thereof as disclosed herein for use in treating or preventing diseases comprising proliferative disorders (such as cancers) , autoimmune diseases, inflammatory disease or infectious diseases.
  • kits or devices and associated methods that employ the antibody or antigen-binding portion thereof as disclosed herein, and pharmaceutical compositions as disclosed herein, which are useful for the treatment of diseases comprising proliferative disorders (such as cancers) , autoimmune diseases, inflammatory disease or infectious diseases.
  • the present invention preferably provides an article of manufacture useful for treating such disorders comprising a receptacle containing the antibody or antigen-binding portion thereof as disclosed herein and instructional materials for using the antibody or antigen-binding portion thereof as disclosed herein to treat, ameliorate or prevent a proliferative disorder or progression or recurrence thereof.
  • Figure 1 is a graph showing antibodies binding to human OX40 transfected CHO-K1 cells.
  • Figure 2 is a graph showing antibodies binding to rhesus monkey OX40 transfected 293F cells.
  • Figure 3 is a graph showing antibodies competitively binding to OX40 with OX40L.
  • Figure 4 is a graph showing antibodies specifically binding to human OX40, but not to human CD40, CD137 and CD271.
  • Figures 5A and 5B are graphs showing epitope binning of the antibodies against benchmark antibodies.
  • Figures 6A, 6B and 6C are graphs showing the effect of antibodies on OX40-stimulated NFkB luciferase activity in Jurkat cells using free antibodies or Fc ⁇ R cross-linking by CD32b-expressing CHO-K1 cells or anti-human IgG Fc reagent. Reporter activity after cross-linking of antibodies by (6A) CD32b-expressing CHO-K1 cells, (6B) native CHO-K1 cells, or (6C) F (ab’) 2 goat anti-human IgG.
  • Figure 7 is a graph showing the effect of antibodies on anti-CD3 induced IL-2 secretion by primary human CD4 + T cells.
  • Figure 8 is a graph showing the effect of antibodies on anti-CD3 induced IFN- ⁇ secretion by primary human CD4 + T cells.
  • Figure 9 is a graph showing the effect of antibodies on anti-CD3 induced proliferation of primary human CD4 + T cells.
  • Figure 10 is a graph showing OX40 expression on activated human CD4 + T cells.
  • Figure 11 is a graph showing the ADCC effect of OX40 antibodies on activated human CD4 + T cells.
  • Figure 12 is a graph showing the CDC effect of OX40 antibodies on activated human CD4 + T cells.
  • antibody or “Ab, ” as used herein, generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions.
  • Light chains of an antibody may be classified into ⁇ and ⁇ light chain.
  • Heavy chains may be classified into ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively.
  • a variable region is linked to a constant region via a “J” region of about 12 or more amino acids, and a heavy chain further comprises a “D” region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ) .
  • a heavy chain constant region consists of 3 domains (C H 1, C H 2 and C H 3) .
  • Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ) .
  • V H and V L region can further be divided into hypervariable regions (called complementary determining regions (CDR) ) , which are interspaced by relatively conservative regions (called framework region (FR) ) .
  • CDR complementary determining regions
  • FR framework region
  • Each V H and V L consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal.
  • the variable region (V H and V L ) of each heavy/light chain pair forms antigen binding sites, respectively. Distribution of amino acids in various regions or domains follows the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype) , IgA1, IgA2, IgD, IgE or IgM antibody.
  • IgG e.g., IgG1, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibody for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype) , IgA1, IgA2, IgD, IgE or IgM antibody.
  • antigen-binding portion or “antigen-binding fragment” of an antibody, which can be interchangeably used in the context of the application, refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody speificaly binds to, and/or compete with the full-length antibody for binding to the same antigen.
  • Antigen binding fragments of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody.
  • antigen binding fragments include Fab, Fab', F (ab') 2 , Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g. scFv) , chimeric antibody, diabody and such polypeptides that comprise at least part of antibody sufficient to confer the specific antigen binding ability on the polypeptides.
  • Antigen binding fragments of an antibody may be obtained from a given antibody (e.g., the monoclonal anti-human OX40 antibody provided in the instant application) by conventional techniques known by a person skilled in the art (e.g., recombinant DNA technique or enzymatic or chemical cleavage methods) , and may be screened for specificity in the same manner by which intact antibodies are screened.
  • a given antibody e.g., the monoclonal anti-human OX40 antibody provided in the instant application
  • conventional techniques known by a person skilled in the art e.g., recombinant DNA technique or enzymatic or chemical cleavage methods
  • monoclonal antibody or “mAb, ” as used herein, refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • recombinant antibody refers to an antibody that is prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
  • anti-OX40 antibody or “OX40 antibody, ” as used herein, refers to an antibody, as defined herein, capable of binding to an OX40 receptor, for example, a human OX40 receptor.
  • OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily.
  • the term “OX40” may include human OX40 receptor, as well as variants, isoforms, and species homologs thereof. Accordingly, an antibody or antigen-binding portion thereof, as defined and disclosed herein, may also bind OX40 from species other than human, for example cynomolgus OX40.
  • human OX40 refers to human sequence OX40, such as the complete amino acid sequence of human OX40 having Genbank Accession No. CAE11757.1.
  • the human OX40 sequence may differ from human OX40 of Genbank Accession No. CAE11757.1 by having, e.g., conserved mutations or mutations in non-conserved regions and the OX40 has substantially the same biological function as the human OX40 of Genbank Accession No. CAE11757.1.
  • mouse OX40 refers to mouse sequence OX40, such as the complete amino acid sequence of mouse OX40 having Genbank Accession No. CAA59476.1.
  • Ka is intended to refer to the association rate of a particular antibody-antigen interaction
  • Kd is intended to refer to the dissociation rate of a particular antibody-antigen interaction.
  • Kd values for antibodies can be determined using methods well established in the art.
  • K D is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) .
  • a preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a system.
  • high affinity for an IgG antibody refers to an antibody having a K D of 1 x 10 -7 M or less, more preferably 5 x 10 -8 M or less, even more preferably 1x10 -8 M or less, even more preferably 5 x 10 -9 M or less and even more preferably 1 x 10 -9 M or less for a target antigen, for example, an OX40 receptor.
  • EC 50 as used herein, which is also termed as “half maximal effective concentration” refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. In the context of the application, EC 50 is expressed in the unit of “nM” .
  • Compet for binding refers to the interaction of two antibodies in their binding to a binding target.
  • a first antibody competes for binding with a second antibody if binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • the alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody can, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its cognate epitope whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope (s) .
  • inhibitor binding refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding of two molecules (eg, human OX40 and human anti-OX40 antibody) to any detectable level.
  • the binding of the two molecules can be inhibited at least 50%by the antibody or antigen-binding fragment thereof.
  • such an inhibitory effect may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
  • epitope refers to a portion on antigen that an immunoglobulin or antibody specifically binds to. “Epitope” is also known as “antigenic determinant” .
  • Epitope or antigenic determinant generally consists of chemically active surface groups of a molecule such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic.
  • an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique steric conformation, which may be “linear” or “conformational” . See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • isolated refers to a state obtained from natural state by artificial means. If a certain “isolated” substance or component is present in nature, it is possible because its natural environment changes, or the substance is isolated from natural environment, or both. For example, a certain un-isolated polynucleotide or polypeptide naturally exists in a certain living animal body, and the same polynucleotide or polypeptide with a high purity isolated from such a natural state is called isolated polynucleotide or polypeptide.
  • isolated excludes neither the mixed artificial or synthesized substance nor other impure substances that do not affect the activity of the isolated substance.
  • isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds an OX40 protein is substantially free of antibodies that specifically bind antigens other than OX40 proteins) .
  • An isolated antibody that specifically binds a human OX40 protein may, however, have cross-reactivity to other antigens, such as OX40 proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • vector refers to a nucleic acid vehicle which can have a polynucleotide inserted therein.
  • the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector.
  • the vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell.
  • Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as ⁇ phage or M13 phage and animal virus.
  • the animal viruses that can be used as vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno- associated virus, herpes virus (such as herpes simplex virus) , pox virus, baculovirus, papillomavirus, papova virus (such as SV40) .
  • a vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene.
  • a vector may comprise origin of replication.
  • host cell refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest.
  • Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue.
  • the term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.
  • immunogenicity refers to ability of stimulating the formation of specific antibodies or sensitized lymphocytes in organisms. It not only refers to the property of an antigen to stimulate a specific immunocyte to activate, proliferate and differentiate so as to finally generate immunologic effector substance such as antibody and sensitized lymphocyte, but also refers to the specific immune response that antibody or sensitized T lymphocyte can be formed in immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors, properties of an antigen, reactivity of a host, and immunization means.
  • transfection refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include but not limited to lipid transfection and chemical and physical methods such as electroporation. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197. In a specific embodiment of the invention, human OX40 gene was transfected into 293F cells.
  • hybridoma and the term “hybridoma cell line, ” as used herein, may be used interchangeably.
  • hybridoma and the term “hybridoma cell line” as used herein, they also include subclone and progeny cell of hybridoma.
  • SPR or “surface plasmon resonance, ” as used herein, refers to and includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) .
  • BIAcore Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.
  • FACS fluorescence-activated cell sorting
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells Natural Killer cells
  • neutrophils neutrophils
  • macrophages cytotoxic cells
  • the antibodies “arm” the cytotoxic cells and are absolutely required for such killing.
  • the primary cells for mediating ADCC, NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) .
  • an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998) .
  • complement dependent cytotoxicity refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed.
  • subject includes any human or nonhuman animal, preferably humans.
  • cancer refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and initiate a medical condition.
  • treatment refers generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis, prevention
  • treating may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • treatment includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
  • an effective amount refers to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • the “an effective amount, ” when used in connection with treatment of OX40-related diseases or conditions refers to an antibody or antigen-binding portion thereof in an amount or concentration effective to treat the said diseases or conditions.
  • prevention refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.
  • pharmaceutically acceptable means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically is compatible with other ingredients in the formulation, and the physiologically compatible with the recipient.
  • a pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer.
  • the pH adjuster includes, but is not limited to, phosphate buffer;
  • the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80;
  • the ionic strength enhancer includes, but is not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator, which can enhance immune response to an antigen or change the type of immune response in an organism when it is delivered together with the antigen to the organism or is delivered to the organism in advance.
  • adjuvants including, but not limited to, aluminium adjuvants (for example, aluminum hydroxide) , Freund’s adjuvants (for example, Freund’s complete adjuvant and Freund’s incomplete adjuvant) , coryne bacterium parvum, lipopolysaccharide, cytokines, and the like.
  • Freund's adjuvant is the most commonly used adjuvant in animal experiments now.
  • Aluminum hydroxide adjuvant is more commonly used in clinical trials.
  • the invention comprises an isolated antibody or an antigen-binding portion thereof.
  • the “antibody” may include polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof; and derivatives thereof including Fc fusions and other modifications, and any other immune-reactive molecule so long as it exhibits preferential association or binding with a OX40 protein.
  • the term further comprises all classes of antibodies (i.e.
  • the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a humanized monoclonal antibody.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including hybridoma techniques, recombinant techniques, phage display technologies, transgenic animals (e.g., a ) or some combination thereof.
  • monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques such as described in more detail in An, Zhigiang (ed. ) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed. 2009; Shire et. al. (eds. ) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1 st ed.
  • a selected binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also an antibody of this invention.
  • the anti-human OX40 monoclonal antibody is prepared by using hybridoma techniques. Generation of hybridomas is well-known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.
  • Mammalian host cells for expressing the antibodies of the present disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. MoI. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells.
  • another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.
  • the antibodies When recombinant expression vectors encoding the antibody are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • the antibodies of the present disclosure are characterized by particular functional features or properties of the antibodies.
  • the isolated antibody or the antigen-binding portion thereof has one or more of the following properties:
  • cytokine e.g., IL-2 or IFN- ⁇
  • the antibody of the invention binds to both human and monkey OX40 with high affinity.
  • the binding of an antibody of the invention to OX40 can be assessed using one or more techniques well established in the art, for instance, ELISA.
  • the binding specificity of an antibody of the invention can also be determined by monitoring binding of the antibody to cells expressing an OX40 protein, e.g., flow cytometry.
  • an antibody can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human OX40, such as CHO cells that have been transfected to express OX40 on their cell surface.
  • Other suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4 + activated T cells, which express native OX40.
  • the binding of the antibody can be tested in BIAcore binding assays.
  • suitable binding assays include ELISA assays, for example using a recombinant OX40 protein.
  • an antibody of the invention binds to a human OX40 with a K D of 1 x 10 -9 M or less, binds to a human OX40 with a K D of 5 x 10 -10 M or less, binds to a human OX40 with a K D of 2 x 10 -10 M or less, binds to a human OX40 protein with a K D of 1 x 10 -10 M or less, binds to a human OX40 protein with a K D of 5 x 10 -11 M or less, binds to a human OX40 protein with a K D of 3 x 10 -11 M or less, or binds to a human OX40 protein with a K D of 2 x 10 -11 M or less.
  • Anti-OX40 antibodies comprising CDRs with sequence identity to specific sequences
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • HCDRs heavy chain CDRs
  • HCDR2 comprising one of the sequences selected from the group consisting of SEQ ID NOs: 3, 7 and 8;
  • LCDRs light chain CDRs
  • Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as set out above, such as, for example, the Kabat numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “Abysis” website at www. bioinf. org. uk/abs (maintained by A.C.
  • sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See Dr. Andrew C.R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S.
  • the Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein. Unless otherwise indicated, all CDRs set forth herein are derived according to Kabat numbering system.
  • Anti-OX40 antibodies comprising CDRs
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • Anti-OX40 antibodies comprising a heavy chain variable region and a light chain variable region
  • the isolated antibody or the antigen-binding portion thereof comprises:
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 10.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 12.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.
  • the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 12.
  • amino acid sequences of the heavy chain variable region and/or the light chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the respective sequences set forth above.
  • the isolated antibody or the antigen-binding portion thereof may contain conservative substitution or modification of amino acids in the variable regions of the heavy chain and/or light chain. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O’ Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43.
  • conservative substitution refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence.
  • a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue.
  • the families of amino acid residues having similar side chains have been defined in the art.
  • amino acids having alkaline side chains for example, lysine, arginine and histidine
  • amino acids having acidic side chains for example, aspartic acid and glutamic acid
  • amino acids having uncharged polar side chains for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids having nonpolar side chains for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids having ⁇ -branched side chains such as threonine, valine, isoleucine
  • amino acids having aromatic side chains for example, tyrosine, phenylalanine, tryptophan, histidine
  • a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family.
  • Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .
  • epitope or immunogenic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups.
  • epitopes may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule.
  • an antibody is said to specifically bind (or immune-specifically bind or react) an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, an antibody is said to specifically bind an antigen when the equilibrium dissociation constant (K D ) is less than or equal to 10 -6 M or less than or equal to 10 -7 M, more preferably when the e K D is less than or equal to 10 -8 M, and even more preferably when the K D is less than or equal to 10 -9 M.
  • K D equilibrium dissociation constant
  • Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • an antibody epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • an epitope may be associated with, or reside in, one or more regions, domains or motifs of, for example, the OX40 protein.
  • the art-recognized term “motif” will be used in accordance with its common meaning and shall generally refer to a short, conserved region of a protein that is typically ten to twenty contiguous amino acid residues.
  • a desired epitope on an antigen it is possible to generate antibodies to that epitope, e.g., by immunizing with a peptide comprising the epitope using techniques described in the present invention.
  • the generation and characterization of antibodies may elucidate information about desirable epitopes located in specific domains or motifs. From this information, it is then possible to competitively screen antibodies for binding to the same epitope.
  • An approach to achieve this is to conduct competition studies to find antibodies that competitively bind with one another, i.e. the antibodies compete for binding to the antigen.
  • a high throughput process for binning antibodies based upon their cross-competition is described in WO 03/48731.
  • Other methods of binning or domain level or epitope mapping comprising antibody competition or antigen fragment expression on yeast are well known in the art.
  • the term “binning” refers to methods used to group or classify antibodies based on their antigen binding characteristics and competition. While the techniques are useful for defining and categorizing the antibodies of the instant invention, the bins do not always directly correlate with epitopes and such initial determinations of epitope binding may be further refined and confirmed by other art-recognized methodology in the art and as described herein. However, it will be appreciated that empirical assignment of the antibodies to individual bins provides information that may be indicative of the therapeutic potential of the disclosed antibodies.
  • epitope mapping techniques include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248: 443-63) (herein specifically incorporated by reference in its entirety) , or peptide cleavage analysis.
  • methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496) (herein specifically incorporated by reference in its entirety) .
  • the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region and/or the light chain variable region of the isolated antibody as disclosed herein.
  • Nucleic acids of the invention can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library e.g., using phage display techniques
  • a nucleic acid encoding such antibodies can be recovered from the gene library.
  • the isolated nucleic acid encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding nucleic acid to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3) .
  • heavy chain constant regions CH1, CH2 and CH3 .
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al. (1991) , supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgG1 or IgG4 constant region.
  • the isolated nucleic acid encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL-or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term “operatively linked” is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the isolated antibody as disclosed herein.
  • the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and comprises a nucleic acid sequence selected from the group consisting of:
  • (C) a nucleic acid sequence that hybridized under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B) .
  • the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the light chain variable region of the isolated antibody as disclosed herein.
  • the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody comprises a nucleic acid sequence selected from the group consisting of:
  • (C) a nucleic acid sequence that hybridized under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B) .
  • the nucleic acid molecule is consisted of SEQ ID NO: 15 or 17.
  • the nucleic acid molecule share an at least 80% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 15 or 17.
  • the percentage of identity is derived from the degeneracy of the genetic code, and the encoded protein sequences remain unchanged.
  • Exemplary high stringency conditions include hybridization at 45°C in 5X SSPE and 45%formamide, and a final wash at 65°C in 0.1 X SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds. ) , Protocols in Molecular Biology, John Wiley &Sons (1994) , pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al, (Eds. ) , Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989) , pp. 9.47 to 9.51.
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug.
  • additional pharmaceutically active ingredients such as another antibody or a drug.
  • the pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, an antiviral agent, or a vaccine, such that the anti-OX40 antibody enhances the immune response against the vaccine.
  • a pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carriers, an aqueous medium, a non-aqueous medium, an anti-microbial agent, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agent, a chelating agent, a diluent, adjuvant, excipient or a nontoxic auxiliary substance, other known in the art various combinations of components or more.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrin.
  • Suitable anti-oxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole, butylated hydroxy toluene and/or propylgalacte.
  • compositions include one or more anti-oxidants such as methionine, reducing antibody or antigen binding fragment thereof may be oxidized.
  • the oxidation reduction may prevent or reduce a decrease in binding affinity, thereby enhancing antibody stability and extended shelf life.
  • the present invention provides a composition comprising one or more antibodies or antigen binding fragment thereof and one or more anti-oxidants such as methionine.
  • the present invention further provides a variety of methods, wherein an antibody or antigen binding fragment thereof is mixed with one or more anti-oxidants, such as methionine, so that the antibody or antigen binding fragment thereof can be prevented from oxidation, to extend their shelf life and/or increased activity.
  • one or more anti-oxidants such as methionine
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • composition of the invention may be administered in vivo, to a subject in need thereof, by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation.
  • compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
  • the appropriate formulation and route of administration may be selected according to the intended application and therapeutic regimen.
  • Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) , in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate) .
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • the particular dosage regimen, including dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc. ) .
  • Frequency of administration may be determined and adjusted over the course of therapy, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis.
  • the dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity.
  • sustained continuous release formulations of a subject therapeutic composition may be appropriate.
  • appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
  • the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • the antibody or the antigen binding portion thereof of the invention may be administered in various ranges. These include about 5 ⁇ g/kg body weight to about 100 mg/kg body weight per dose; about 50 ⁇ g/kg body weight to about 5 mg/kg body weight per dose; about 100 ⁇ g/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 ⁇ g/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the dosage is at least about 100 ⁇ g/kg body weight, at least about 250 ⁇ g/kg body weight, at least about 750 ⁇ g/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight.
  • the antibody or the antigen binding portion thereof of the invention is preferably administered as needed to subjects in need thereof. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.
  • the course of treatment involving the antibody or the antigen-binding portion thereof of the instant invention will comprise multiple doses of the selected drug product over a period of weeks or months. More specifically, the antibody or the antigen-binding portion thereof of the instant invention may be administered once every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it will be appreciated that the dosages may be altered or the interval may be adjusted based on patient response and clinical practices.
  • Dosages and regimens may also be determined empirically for the disclosed therapeutic compositions in individuals who have been given one or more administration (s) .
  • individuals may be given incremental dosages of a therapeutic composition produced as described herein.
  • the dosage may be gradually increased or reduced or attenuated based respectively on empirically determined or observed side effects or toxicity.
  • a marker of the specific disease, disorder or condition can be followed as described previously.
  • these include direct measurements of tumor size via palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; an improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; the measurement of an indirect tumor marker (e.g., PSA for prostate cancer) or a tumorigenic antigen identified according to the methods described herein, a decrease in pain or paralysis; improved speech, vision, breathing or other disability associated with the tumor; increased appetite; or an increase in quality of life as measured by accepted tests or prolongation of survival.
  • an indirect tumor marker e.g., PSA for prostate cancer
  • the dosage will vary depending on the individual, the type of neoplastic condition, the stage of neoplastic condition, whether the neoplastic condition has begun to metastasize to other location in the individual, and the past and concurrent treatments being used.
  • Compatible formulations for parenteral administration will comprise the antibody or antigen-binding portion thereof as disclosed herein in concentrations of from about 10 ⁇ g/ml to about 100 mg/ml.
  • the concentrations of the antibody or the antigen binding portion thereof will comprise 20 ⁇ g/ml, 40 ⁇ g/ml, 60 ⁇ g/ml, 80 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml, 300, ⁇ g/ml, 400 ⁇ g/ml, 500 ⁇ g/ml, 600 ⁇ g/ml, 700 ⁇ g/ml, 800 ⁇ g/ml, 900 ⁇ g/ml or 1 mg/ml.
  • the concentrations of the antibody or the antigen binding portion thereof will comprise 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml
  • the antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, detection of OX40 or enhancement of immune response.
  • these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations.
  • the immune response can be modulated, for instance, augmented, stimulated or up-regulated.
  • the subjects include human patients in need of enhancement of an immune response.
  • the methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., the T-cell mediated immune response) .
  • the methods are particularly suitable for treatment of cancer in vivo.
  • the anti-OX40 antibodies can be administered together with an antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject) .
  • the two can be administered in either order or simultaneously.
  • the invention further provides methods for detecting the presence of human OX40 antigen in a sample, or measuring the amount of human OX40 antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to human OX40, under conditions that allow for formation of a complex between the antibody or portion thereof and human OX40. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative of the presence of human OX40 antigen in the sample.
  • the anti- OX40 antibodies of the invention can be used to purify human OX40 via immunoaffinity purification.
  • the present invention provides a method of treating a disorder in a mammal, which comprises administering to the subject (for example, a human) in need of treatment a therapeutically effective amount of the antibody or antigen-binding portion thereof as disclosed herein.
  • the disorder is a cancer.
  • cancers where OX40 is implicated may be treated or prevented with a method provided by the disclosure.
  • the cancers may be solid cancers or hematologic malignancies.
  • lung cancers such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma) , alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous) , and sarcoma (cancerous) ; heart cancer such as myxoma, fibromas, and rhabdomyomas; bone cancers such as osteochondromas, condromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcomas, malignant fibrous his
  • bronchogenic carcinoma e.g., squam
  • examples of cancer include but not limited to B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL) ; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL) ; acute lymphoblastic leukemia (ALL) ; Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliierative disorder (PTLD) , as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) , B-cell proliferative disorders, and Meigs’ syndrome.
  • B-cell lymphoma including low grade/f
  • More specific examples include, but are not limited to, relapsed or refractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicle center lymphoma (folli
  • examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B-Cell Non-Hodgkin’s lymphomas (NHL) ) and lymphocytic leukemias.
  • lymphomas e.g., B-Cell Non-Hodgkin’s lymphomas (NHL)
  • lymphocytic leukemias include e.g.
  • follicular lymphomas a) follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt’s lymphoma (including endemic Burkitt’s lymphoma, sporadic Burkitt’s lymphoma and Non-Burkitt’s lymphoma) , c) marginal zone lymphomas (including extranodal marginal zone B-cell lymphoma (Mucosa-associated lymphatic tissue lymphomas, MALT) , nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma) , d) Mantle cell lymphoma (MCL) , e) Large Cell Lymphoma (including B-cell diffuse large cell lymphoma (DLCL) , Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-Cell Lymp
  • the disorder is an autoimmune disease.
  • autoimmune diseases that may be treated with the antibody or antigen-binding portion thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis.
  • the antibody or the antigen-binding portion thereof may also be used to treat or prevent infectious disease, inflammatory disease (such as allergic asthma) and chronic graft-versus-host disease.
  • the invention also provides a method of enhancing (for example, stimulating) an immune response in a subject comprising administering an antibody or an antigen binding portion thereof of the invention to the subject such that an immune response in the subject is enhanced.
  • the subject is a mammal. In a specific embodiment, the subject is a human.
  • the term “enhancing an immune response” or its grammatical variations, means stimulating, evoking, increasing, improving, or augmenting any response of a mammal’s immune system.
  • the immune response may be a cellular response (i.e. cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (i.e. antibody mediated response) , and may be a primary or secondary immune response.
  • Examples of enhancement of immune response include increased CD4 + helper T cell activity and generation of cytolytic T cells.
  • the enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production or IFN- ⁇ production) , regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.
  • cytotoxic T lymphocyte assays release of cytokines (for example IL-2 production or IFN- ⁇ production) , regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.
  • methods of the disclosure enhance the immune response by a mammal when compared to the immune response by an untreated mammal or a mammal not treated using the methods as disclosed herein.
  • the antibody or an antigen binding portion thereof is used to enhance the immune response of a human to a microbial pathogen (such as a virus) .
  • the antibody or an antigen binding portion thereof is used to enhance the immune response of a human to a vaccine.
  • the method enhances a cellular immune response, particularly a cytotoxic T cell response.
  • the cellular immune response is a T helper cell response.
  • the immune response is a cytokine production, particularly IFN- ⁇ production or IL-2 production.
  • the antibody or an antigen binding portion thereof may be used to enhance the immune response of a human to a microbial pathogen (such as a virus) or to a vaccine.
  • the antibody or the antigen-binding portion thereof may be used alone as a monotherapy, or may be used in combination with chemical therapies or radiotherapies.
  • the antibody or the antigen-binding portion thereof may be used in combination with an anti-cancer agent, a cytotoxic agent or chemotherapeutic agent.
  • anti-cancer agent or “anti-proliferative agent” means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that, in selected embodiments as discussed above, such anti-cancer agents may comprise conjugates and may be associated with the disclosed site-specific antibodies prior to administration.
  • selected anti-cancer agents will be linked to the unpaired cysteines of the engineered antibodies to provide engineered conjugates as set forth herein. Accordingly, such engineered conjugates are expressly contemplated as being within the scope of the instant invention. In other embodiments, the disclosed anti-cancer agents will be given in combination with site-specific conjugates comprising a different therapeutic agent as set forth above.
  • cytotoxic agent means a substance that is toxic to the cells and decreases or inhibits the function of cells and/or causes destruction of cells.
  • the substance is a naturally occurring molecule derived from a living organism.
  • cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A) , fungal (e.g., ⁇ -sarcin, restrictocin) , plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S)
  • chemotherapeutic agent comprises a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents) .
  • cancer cells e.g., cytotoxic or cytostatic agents
  • Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly.
  • vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis.
  • chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous or generate tumorigenic progeny (e.g., TIC) .
  • Such agents are often administered, and are often most effective, in combination, e.g., in regimens such as CHOP or FOLFIRI.
  • anti-cancer agents that may be used in combination with the site-specific constructs of the present invention (either as a component of a site specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, a camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens and selective estrogen receptor modulators aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens
  • troxacitabine a 1, 3-dioxolane nucleoside cytosine analog
  • antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor
  • vaccines rIL-2; topoisomerase 1 inhibitor; rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the present invention also provides for the combination of the antibody or the antigen-binding portion thereof with radiotherapy (i.e., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like) .
  • radiotherapy i.e., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like
  • Combination therapy using the directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates may be used in connection with a targeted anti-cancer agent or other targeting means.
  • radiation therapy is administered in pulses over a period of time from about 1 to about 2 weeks.
  • the radiation therapy may be administered to subjects having head and neck cancer for about 6 to 7 weeks.
  • the radiation therapy may be administered as a single dose or as multiple, sequential doses.
  • the invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from a patient to identify tumor cells including tumorigenic cells.
  • Such methods include identifying an individual having cancer for treatment or monitoring progression of a cancer, comprising contacting the patient or a sample obtained from a patient (either in vivo or in vitro) with an antibody as described herein and detecting presence or absence, or level of association, of the antibody to bound or free target molecules in the sample.
  • the antibody will comprise a detectable label or reporter molecule as described herein.
  • the association of the antibody with particular cells in the sample can denote that the sample may contain tumorigenic cells, thereby indicating that the individual having cancer may be effectively treated with an antibody as described herein.
  • Samples can be analyzed by numerous assays, for example, radioimmunoassays, enzyme immunoassays (e.g. ELISA) , competitive-binding assays, fluorescent immunoassays, immunoblot assays, Western Blot analysis and flow cytometry assays.
  • Compatible in vivo theragnostic or diagnostic assays can comprise art recognized imaging or monitoring techniques, for example, magnetic resonance imaging, computerized tomography (e.g. CAT scan) , positron tomography (e.g., PET scan) , radiography, ultrasound, etc., as would be known by those skilled in the art.
  • a unit dosage comprising one or more containers, comprising one or more doses of the antibody or the antigen-binding portion thereof are also provided.
  • a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising, for example, the antibody or the antigen-binding portion thereof, with or without one or more additional agents.
  • such a unit dosage is supplied in single-use prefilled syringe for injection.
  • the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range.
  • the conjugate composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water or saline solution.
  • the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on, or associated with, the container (s) indicates that the enclosed conjugate composition is used for treating the neoplastic disease condition of choice.
  • kits for producing single-dose or multi-dose administration units of site-specific conjugates and, optionally, one or more anti-cancer agents comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed conjugates in a conjugated or unconjugated form.
  • the container (s) comprise a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • kits will generally contain in a suitable container a pharmaceutically acceptable formulation of the engineered conjugate and, optionally, one or more anti-cancer agents in the same or different containers.
  • the kits may also contain other pharmaceutically acceptable formulations, either for diagnosis or combined therapy.
  • such kits may contain any one or more of a range of anti-cancer agents such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and/or other anti-cancer agents.
  • kits may have a single container that contains the disclosed the antibody or the antigen-binding portion thereof, with or without additional components, or they may have distinct containers for each desired agent.
  • a single solution may be pre-mixed, either in a molar equivalent combination, or with one component in excess of the other.
  • the conjugates and any optional anti-cancer agent of the kit may be maintained separately within distinct containers prior to administration to a patient.
  • kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline (PBS) , Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • PBS phosphate-buffered saline
  • Ringer's solution dextrose solution
  • the liquid solution is preferably an aqueous solution, with a sterile aqueous or saline solution being particularly preferred.
  • the components of the kit may be provided as dried powder (s) .
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
  • kits may also contain a means by which to administer the antibody or the antigen-binding portion thereof and any optional components to a patient, e.g., one or more needles, I. V. bags or syringes, or even an eye dropper, pipette, or other such like apparatus, from which the formulation may be injected or introduced into the animal or applied to a diseased area of the body.
  • the kits of the present invention will also typically include a means for containing the vials, or such like, and other component in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.
  • cDNA encoding the extracellular domain (ECD) of OX40 protein (Gen Bank ref CAB96543.1) were synthesized by Sangon Biotech and inserted into a modified expression vector pcDNA3.3 (ThermoFisher) . Max-prep the plasmid DNAs and the inserted DNA sequences were verified by sequencing. Fusion proteins OX40 ECD conjugated with human Fc or His tag were obtained by transfection of human OX40 ECD gene into Freestyle 293F (ThermoFisher) or Expi-293F cells (ThermoFisher) . After 5 days, supernatants were harvested from the cultures of transient transfected cells. The fusion proteins were purified and quantitated for usage of immunization and screening.
  • WBP341-BMK1 also referred to as “BMK1”
  • WBP341-BMK5 also referred to as “BMK5”
  • WBP341-BMK7 also referred to as “BMK7”
  • WBP341-BMK10 also referred to as “BMK10”
  • WBP341-BMK1 was synthesized according to the clone of 11D4 from U.S. Patent No. US8236930B2 (Pfizer)
  • WBP341-BMK5 was synthesized according to the clone of 106-22 from U.S. Patent Application No.
  • WBP341-BMK7 was synthesized according to the clone of OX40mAb24 from PCT publication No. WO2016057667 (MedImmune) .
  • WBP341-BMK10 was synthesized according to the clone of 1A7. gr1 from PCT publication No. WO2015153513 (Genentech) .
  • OX40 transfectant cell lines In order to obtain tools for antibody screening and validation, we generated OX40 transfectant cell lines. Briefly, CHO-K1 or 293F cells were transfected with the modified expression vector pcDNA3.3 containing full-length OX40 using Lipofectamine 2000 or PlasFect transfection kit according to manufacturer’s protocol. At 48 -72 hours post transfection, the transfected cells were cultured in medium containing Blasticidin for selection. Overtime this will select the cells that have the expression plasmid stably incorporated into their genomic DNAs. Meanwhile the cells were checked for OX40 expression. Once the expression verified, single clones of interested were picked by limited dilution and scaled up to large volumes. The established monoclonal cell lines were then maintained in medium containing Blasticidin.
  • mice To generate mouse anti-human OX40 monoclonal antibodies, Balb/c mice, 6-8 weeks of age, were immunized with 20 ⁇ g of human OX40 ECD protein in aluminium phosphate (Alum-Phos) in footpad and 20 ⁇ g of human OX40 ECD protein in TiterMax subcutaneously for first boost, and repeat the immunization every two weeks with human OX40 ECD protein in Alum-Phos or TiterMax.
  • the serum antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) every three or four weeks. When the serum antibody titer was sufficiently high, the mice were given a final boost with 40 ⁇ g of human OX40 ECD protein in PBS without adjuvant.
  • ELISA enzyme-linked immunosorbent assay
  • the cell fusion was performed as following: preparing myeloma cells SP2/0, myeloma cells were thawed the week before the fusion, and were split 1: 2 each day until the day before the fusion to keep the cells in logarithmic growth phase.
  • B lymphocytes isolated from lymph node and spleen of immunized mice were combined with myeloma cells (at 1: 1.1 ratio) .
  • Cell mixture was washed and re-suspended in ECF solution at 2 ⁇ 10 6 cells/mL. The cells are ready for ECF.
  • ECF solution After electronic cell fusion, cell suspension from the fusion chamber was immediately transferred into a sterile tube containing more medium, and incubated for 2 hours in a 37 °C incubator.
  • the cell suspension was mixed and transferred into 96-well plates (1 ⁇ 10 4 cells/well) .
  • the 96-well plates were cultured at 37°C, 5%CO 2 , and were monitored periodically. When the clones were big enough, 100 uL of supernatant were transferred from the tissue culture plates to 96-well assay plates for antibody screening.
  • ELISA was used as first screening method to test the binding of hybridoma supernatants to human OX40 protein. Briefly, plates (Nunc) were coated with human OX40 ECD overnight at 4 °C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 2 hours. The plates were then washed and subsequently incubated with secondary antibody, goat anti-mouse IgG HRP (Bethyl) , for 1 hour. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device) .
  • CHO-K1 cells expressing human OX40 were transferred into 96-well U-bottom plates (Corning) at a density of 1 ⁇ 10 5 cells/well. The hybridoma supernatants were then loaded to the cells and incubated for 1 h at 4 °C. After washing with 1 ⁇ PBS/1%BSA, the secondary antibody goat anti-mouse IgG Fc Alexa647 (Jackson ImmunoResearch Lab) was applied and incubated with cells at 4 °C in the dark for half an hour. The cells were then washed and resuspended in 1 ⁇ PBS/1%BSA or fixed with 4%paraformldehyde, and analyzed by flow cytometry (BD) .
  • BD flow cytometry
  • Antibody binding to parental CHO-K1 cell line was used as negative control.
  • the binding of hybridoma supernatants to mouse and monkey OX40 protein was tested using ELISA as a second round of screening. Briefly, plates (Nunc) were coated with mouse or cynomolgus monkey OX40 ECD protein overnight at 4 °C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 2 hours. The plates were then washed and subsequently incubated with secondary antibody, goat anti-mouse IgG HRP (Bethyl) , for 1 hour. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader.
  • human OX40/CD40 fusion protein-overexpressing Jurkat NFkB-luciferase reporter cell was constructed as described above. The cells were cultured in complete RPMI1640 medium containing 10%FBS, and 0.5 mg/mL of Hygromycin B as selection. OX40 Jurkat reporter cells were collected and added to a 96-well plate at 4 ⁇ 10 4 cells/well.
  • the positive hybridoma cell lines were used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to give 180 cells per 1.5 mL semi-solid medium. The cells were mixed gently in vortex oscillators for 5-10 seconds and then seeded in 6-well plates. The plates were kept in a 37°C, 5%CO 2 incubator for 7-8 days. Each visible single colony was picked into 96-well plates with DMEM medium supplemented with 10%FBS. After 2-3 days’ culture, the cell supernatants were collected and checked by binding and functional screening. The exhausted supernatant (ESN) of selected single clones were collected, and the antibodies were purified for further characterization.
  • ESN exhausted supernatant
  • RNAs were isolated from monoclonal hybridoma cells using RNeasy Plus Mini Kit (Qiagen) with Trizol reagent.
  • the heavy chain variable region (VH) and heavy chain variable region (VL) of OX40 chimeric antibodies were amplified as follows. Briefly, RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here.
  • Step1 Step2 Step3 Step4 Temperature (°C) 25 37 85 4 Time 10 min 120 min 5 min ⁇
  • the resulting cDNA was used as template for subsequent PCR amplification using primers specific for interested genes.
  • the PCR reaction was done as follows.
  • Component Amount cDNA 1 ⁇ L Ex PCR buffer 5 ⁇ L dNTP 2 ⁇ L ExTaq 0.5 ⁇ L P1 (25pM) 0.5 ⁇ L P2 (25pM) 0.5 ⁇ L ddH 2 O 40.5 ⁇ L
  • the PCR product (10 ⁇ L) was inserted into the pMD18-T vector; and 10 ⁇ L of the ligation product was transformed into the Top 10 competent cells. Transformed cells were plated on 2-YT+Cab plates and incubated overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers followed by sequencing.
  • Hybridoma antibodies with high affinity and specificity to OX40 were selected for humanization, in order to reduce the risk of immunogenicity when used in clinical trials.
  • the VH and VL genes of hybridoma antibodies were re-amplified with cloning primers containing appropriate restriction sites and cloned into a modified human IgG1 expression vector to create corresponding clones of chimeric antibody.
  • “Best Fit” approach was used to humanize the antibodies’ light and heavy chains. For light chains, amino acid sequences of corresponding V-genes were blasted against in-house human germline V-gene database. The sequence of humanized VL-gene was derived by replacing human CDR sequences in the top hit with mouse CDR sequences using Kabat CDR definition.
  • the parental clone W3411-1.61.7 was constructed as chimeric form comprising variable region of W3411-1.61.7 and constant region of human IgG1, named as W3411-1.61.7-x1-IgG1K.
  • the human germline acceptor template chosen for the VH framework 1-3 was IGHV3-7*01 and for the VL framework 1-3 was IGKV7-3*01.
  • Antibody sequence optimization was carried out by introducing appropriate modification at specific site into the nucleotide sequence encoding an antibody.
  • Human-to-mouse back mutations were introduced into humanized VH and VL genes by side directed mutagenesis. Specific amino acid substitutions were selected based on structural modeling of original mouse and humanized antibodies and using information from previous humanization projects.
  • PTM post-translational modification site removing mutations were introduced by site directed mutagenesis using QuickChange mutagenesis kit according to the manufacturer’s protocol.
  • the amino acid NYT in CDR2 of heavy chain was identified as a glycosylation site, so antisense mutagenic nucleotides were designed to introduce following mutations: N to Q (NYT-QYT) , N to S (NYT-SYT) or T to A (NYT-QYA) . All mutations were verified by sequencing.
  • VH genes and VL genes of humanized OX40 antibody were synthesized by Genewiz, and then cloned into the modified human IgG1 expression vector pcDNA3.4 (ThermoFisher) .
  • Expi-293F cells were transiently transfected with the vector for antibody expression. The culture supernatant containing antibodies was harvested and purified using Protein A chromatography.
  • Antibodies were characterized for affinity and binding kinetics to OX40 by SPR assay using Biacore T200 (GE) .
  • Anti-human IgG antibody was pre-immobilized to a sensor chip (CM5) , and anti-OX40 antibodies in running buffer (1 ⁇ HBS-EP+, GE) were captured when injected to the chip. Then various concentrations of human or monkey OX40 and running buffer were flowed through the sensor chip at a flow rate of 30 ⁇ L/min for an association phase of 180 s, followed by dissociation.
  • the association and dissociation curve was fit by 1: 1 Langmuir binding model using the BIAevaluation T200 Software Version2.0.
  • the illustrative antibodies of the present disclosure including W3411-1.61.7-z17-p1-IgG1K and W3411-1.61.7-z19-p1-IgG1K bound to human OX40 with high specificity, with a K D of 4.09 ⁇ 10 -10 and 6.54 ⁇ 10 -11 , respectively.
  • the illustrative antibodies of the present disclosure including W3411-1.61.7-z17-p1-IgG1K and W3411-1.61.7-z19-p1-IgG1K bound to monkey OX40 with high specificity, with a K D of 1.19 ⁇ 10 -8 and 2.27 ⁇ 10 -9 , respectively.
  • Cell-based FACS was used for testing the binding activity of anti-OX40 antibodies to OX40.
  • Human OX40-expressing CHO-K1 cells were transferred into 96-well U-bottom plates (Corning) at a density of 1 ⁇ 10 5 cells/well. Testing antibodies were serially diluted in wash buffer (1 ⁇ PBS/1%BSA) and incubated with cells at 4 °C for 1 h. After washing with 1 ⁇ PBS/1%BSA, the secondary antibody goat anti-human IgG Fc-PE (Jackson ImmunoResearch Lab) was applied and incubated with cells at 4 °C in the dark for 1 h. The cells were then washed and resuspended in 1 ⁇ PBS/1%BSA or fixed with 4%paraformldehyde, and then analyzed by flow cytometry (BD) .
  • BD flow cytometry
  • Figure 1 shows the binding of the antibodies to human OX40 transfected CHO-K1 cells.
  • ELISA based competition assay was used to test whether anti-OX40 antibodies could competitively block the binding of OX40 to OX40 ligand (OX40L) . Briefly, plates (Nunc) were coated with human OX40 ECD overnight at 4 °C. Antibodies were serially diluted in blocking buffer and mixed with constant concentration of OX40L. After blocking and washing, the antibody/OX40L mixture were added to the plates, and then incubated at room temperature for 1 h. The plates were then washed and subsequently incubated with HRP conjugated secondary antibody for 1 h to detect the binding of OX40L to OX40 ECD. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device) .
  • Cross-reactivity of anti-OX40 antibodies to rhesus monkey OX40 was measured by cell-based FACS. Briefly, rhesus monkey OX40-expressing 293F cells were transferred into 96-well U-bottom plates (Corning) at a density of 2 ⁇ 10 5 cells/well. Testing antibodies were serially diluted in wash buffer (1 ⁇ PBS/1%BSA) and incubated with cells at 4 °C for 1 h. After washing with 1 ⁇ PBS/1%BSA, the secondary antibody goat anti-human IgG Fc-PE (Jackson ImmunoResearch Lab) was applied and incubated with cells at 4 °C in the dark for 1 h. The cells were then washed and resuspended in 1 ⁇ PBS/1%BSA or fixed with 4%paraformldehyde, and then analyzed by flow cytometry (BD) .
  • BD flow cytometry
  • Figure 2 shows the binding of the antibodies to rhesus monkey OX40 transfected 293F cells.
  • the EC50 of antibodies binding activity to cell-surface human and monkey OX40 is shown in Table 9 below.
  • Human OX40, CD40, 4-1BB (CD137) , GITR (CD357) and CD271 ECD were coated on plates (Nunc) overnight at 4 °C. After blocking and washing, testing antibodies were diluted in blocking buffer and added to the plates and incubated at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody goat anti-human IgG Fc-HRP (Bethyl) for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device) .
  • the antibodies “W3411-1.61.7-z17-p1-IgG1K” and “W3411-1.61.7-z19-p1-IgG1K” specifically bind to human OX40, but not to human CD40, CD137, CD271 and CD357.
  • the binding epitope of anti-OX40 antibodies was binned against benchmark antibodies by ELISA.
  • the testing antibodies were coated on plates (Nunc) overnight at 4 °C. After blocking and washing, constant concentration of human OX40 protein diluted in blocking buffer was added to the plates and incubated at room temperature for 1 h. Then the biotinylated benchmarks were serially diluted and added to each well and incubated for another 1 h. The plates were then washed and subsequently incubated with secondary antibody steptavidin-HRP (Life Technology) for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device) .
  • anti-OX40 antibodies to signal through human OX40 was assessed using an engineered Jurkat cell line expressing OX40/CD40 fusion protein and NFkB-luciferase reporter gene. Bioactivity of anti-OX40 antibodies cross-linked using an anti-human IgG Fc reagent or cells expressing human Fc ⁇ receptor complements were measured.
  • the Jurkat NFkB-luciferase Reporter cells were cultured in complete RPMI 1640 medium containing 10%FBS, and 0.5 mg/mL of Hygromycin B as selection.
  • CD32b-expressing CHO-K1 cells or F (ab’) 2 goat anti-human IgG was used to mediate antibodies cross-linking, which clusters and activates OX40 on the Jurkat report cells.
  • OX40 Jurkat reporter cells were collected and added to a 96-well plate.
  • OX40 antibodies serially diluted in complete medium were added to the cells in the presence of CD32b-expressing CHO-K1 cells, parental CHO-K1 cells or cross-linker antibodies, and incubated the plates at 37 °C, 5%CO 2 for 6 hours or overnight.
  • Reconstituted luciferase substrate Promega was added to each well and mixed well. The luciferase intensity was read using a microplate reader (Molecular Device) .
  • Anti-OX40 antibodies were also tested for bioactivity in soluble condition.
  • Human CD4 + T cells used in this example were isolated from human PBMCs using Human CD4 + T Cell Enrichment Kit (StemCell) according to the manufacturer’s protocol. The cells were resuspended in complete RPMI 1640 medium.
  • non-tissue culture treated flat-bottom 96-well plates (Corning) were pre-coated with anti-CD3 overnight at 4 °C.
  • the plates were washed with complete RPMI 1640 medium to remove un-bound antibodies.
  • Freshly isolated human CD4 + T cells were added to each well at a density of 1 ⁇ 10 5 cells/well in a volume of 100 ⁇ L.
  • constant concentration of cross linking antibody F (ab’) 2 goat anti-human IgG and serially diluted OX40 antibodies were mixed in 100 ⁇ L and were also added to each well of the plates.
  • the plates were incubated at 37 °C, 5%CO 2 for 3 days and then the supernatants were harvested for IL-2 measurement by ELISA.
  • an assay to co-stimulate human CD4 + T cells through OX40 signal in combination with CD3/T cell receptor (TCR) complex was performed. Briefly, non-tissue culture treated flat-bottom 96-well plates (Corning) were pre-coated with 100 ⁇ L of mixture of constant concentration of anti-CD3 and different concentration of anti-OX40 antibodies. The plates were incubated overnight at 4 °C, and then washed with complete RPMI 1640 medium to remove un-bound antibodies. Freshly isolated human CD4 + T cells were added to each well at a density of 1 ⁇ 10 5 cells/well in a volume of 200 ⁇ L.
  • the plates were incubated at 37 °C, 5%CO 2 for 3 days and then the supernatants were harvested for IFN ⁇ measurement by ELISA.
  • the cell pellets were harvested to measure CD4 + T cell proliferation by CellTiter-Glo (Promega) .
  • the luminescence was read using a microplate reader (Molecular Device) .
  • OX40 is expressed on variety of cell types. In order to assess their ability to trigger Fc effector function, the anti-OX40 antibodies were evaluated whether they could induce ADCC and CDC effect on OX40 expressing cells.
  • PMA and Ionomycin activated human CD4 + T cells, as target, and various concentrations of anti-OX40 antibodies were pre-incubated in 96-well round-bottom plate (BD) for 30 minutes; and then allogeneic PBMCs, as effector, were added at effector/target ratio of 50: 1.
  • the plate was kept at 37 °C, 5%CO 2 for 6 hours.
  • Target cell lysis was determined by LDH-based Cytotoxicity Detection Kit (Roche) .
  • the absorbance at 492nm was read using a microplate reader (Molecular Device) .
  • OX40 antibodies specifically bind to human OX40, without cross-reactivity to mouse OX40 and human CD40, despite sharing 60%and 23%identity in their aa sequence respectively.
  • twenty-two variants were constructed by replacing the following residues of the extracellular domain of human OX40 (hPro1) with the corresponding mouse OX40 (mPro1) amino acid (referred to as “aa” in brief hereinafter) or human CD40 aa (hPro40) .
  • FL-x1 also referred as “x1” hereinafter
  • CRDmox40_1 Human OX40 aa 29 to 65 replace with the mouse counterparts
  • FL-x3 also referred as “x3” hereinafter
  • CRDmox40_3 Human OX40 aa 108 to 146 replace with the mouse counterparts
  • FL-x4 also referred as “x4” hereinafter
  • CRDmox40_4 Human OX40 aa 147 to 214 replace with the mouse counterparts
  • FL-x5 also referred as “x5” hereinafter
  • CRDmox40_1-2 Human OX40 aa 29 to 107 replace with the mouse counterparts
  • FL-x6 also referred as “x6” hereinafter
  • CRDmox40_2-3 Human OX40 aa 66 to 146 replace with the mouse counterparts
  • FL-x7 also referred as “x7” hereinafter
  • CRDmox40_3-4 Human OX40 aa 108 to 214 replace with the mouse counterparts
  • FL-x8 also referred as “x8” hereinafter: CRDmox40_1-3 (Human OX40 aa 29 to 146 replace with the mouse counterparts)
  • FL-x9 also referred as “x9” hereinafter
  • CRDmox40_2-4 Human OX40 aa 66 to 214 replace with the mouse counterparts
  • FL-x10 also referred as “x10” hereinafter
  • CRDmox40_1, 2, 4 Human OX40 aa 29 to 107 and 147 to 214 replace with the mouse counterparts
  • FL-x11 also referred as “x11” hereinafter
  • CRDmox40_1, 3, 4 Human OX40 aa 29 to 65 and 108 to 214 replace with the mouse counterparts
  • FL-x12 also referred as “x12” hereinafter
  • CRDhcd40_1 Human OX40 aa 29 to 65 replace with the human CD40 aa counterparts
  • FL-x13 also referred as “x13” hereinafter
  • CRDhcd40_2 Human OX40 aa 66 to 107 replace with the human CD40 aa counterparts
  • FL-x14 also referred as “x14” hereinafter
  • CRDhcd40_3 Human OX40 aa 108 to 146 replace with the human CD40 aa counterparts
  • FL-x15 also referred as “x15” hereinafter
  • CRD hcd40_4 Human OX40 aa 147 to 214 replace with the human CD40 aa counterparts
  • FL-x16 also referred as “x16” hereinafter
  • CRDhcd40_1-2 Human OX40 aa 29 to 107 replace with the human CD40 aa counterparts
  • FL-x17 also referred as “x17” hereinafter
  • CRDhcd40_2-3 Human OX40 aa 66 to 146 replace with the human CD40 aa counterparts
  • FL-x18 also referred as “x18” hereinafter
  • CRDhcd40_3-4 Human OX40 aa 108 to 214 replace with the human CD40 aa counterparts
  • FL-x19 also referred as “x19” hereinafter: CRDhcd40_1-3 (Human OX40 aa 29 to 146 replace with the human CD40 aa counterparts)
  • FL-x20 also referred as “x20” hereinafter
  • CRDhcd40_2-4 Human OX40 aa 66 to 214 replace with the human CD40 aa counterparts
  • FL-x21 also referred as “x21” hereinafter: CRDhcd40_1, 2, 4 (Human OX40 aa 29 to 107 and 147 to 214 replace with the human CD40 aa counterparts)
  • FL-x22 also referred as “x22” hereinafter: CRDhcd40_1, 3, 4 (Human OX40 aa 29 to 65 and 108 to 214 replace with the human CD40 aa counterparts)
  • the twenty-two variants were cloned into pcDNA3.0 vector, and used for 293F cells transfection. Briefly, 293F cells were diluted to a density of 1 ⁇ 10 6 cells/mL with FreeStyle 293F medium and aliquots of 3 mL per well were added to 24-well plate. Transfections were performed using 293fectin reagent (Life Technologies) . For each transfection, 3 ⁇ g of DNA were diluted in 150 ⁇ L Opti-MEMI reduced serum medium (life Technologies) , and then combined with 6 ⁇ L 293fectin reagent pre-diluted in 150 ⁇ L Opti-MEMI reduced serum medium. The DNA/Lipofectamine mixture was allowed to stand at 25 °C for 20 min before being added to the culture. The transfected cells were analyzed by flow cytometry 48h post-transfection.
  • Binding of antibodies to chimeric OX40 variants was analyzed by flow cytometry. Briefly, 1 ⁇ g/mL antibodies, except BMK10 is 2 ⁇ g/mL, were incubated with chimeric OX40 expressed transfected 293F cells for 1 hour at 4 °C, and then incubated with 3 ⁇ g/mL goat anti-human IgG Fc R-PE (Jackson ImmunoResearch Lab) for 40 min at 4 °C. Cells were analyzed using flow cytometer.
  • Table 13 shows the domain of OX40 (colored in grey) involved in the antigen binding.
  • CCD1 refers to amino acids 29-65 of human OX4
  • CCD2 refers to amino acids 66-107 of human OX4
  • CCD3 refers to amino acids 108-146 of human OX4
  • CCD4 refers to amino acids 147-214 of human OX4.
  • Figures 5A and 5B show the results of epitope binning of the antibodies “W3411-1.61.7-z17-p1-IgG1K, ” and “W3411-1.61.7-z19-p1-IgG1K” against benchmark antibodies BMK5, BMK7 and BMK10.
  • His plasmid that encodes ECD of human OX40 and a C-terminal His-tag was used as template, and a set of mutagenic primers was used for the first step PCR using the QuikChange lightning multi-site-directed mutagenesis kit (Agilent technologies, Palo Alto, CA) . Dpn I endonuclease was used to digest the parental template after mutant strand synthesis reaction.
  • linear DNA expression cassette which composed of CMV promoter, an extracellular domain of OX40, a His-tag and a herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in 293F cells at 37 °C (Life Technologies, Gaithersburg, MD) , quantified by His-tag quantification ELISA.
  • TK herpes simplex virus thymidine kinase
  • Monoclonal antibody W3411-1.61.7-z19-p1-IgG1K (2 ⁇ g/mL) was coated in plates for ELISA binding assay. After interacting with the supernatant containing quantified OX40 mutants or human OX40-ECD. His protein, HRP conjugated anti-His antibody (1: 5000, GenScript-A00612, CHN) was added as detection antibody. Absorbance was normalized according to the average of control mutants. After setting an additional cutoff to the binding fold change ( ⁇ 0.75) , the final determined epitope residues were identified by considering domain mapping, epitope mapping and crystal structure, which did not include the amino acids contributing to structure stability, such as amino acids belonging to CRD3 &CRD4.
  • the normalized fold change of OX40 point mutations on antibody binding was shown in Table 14. Hotspots were identified by considering domain mapping, alanine scanning (cutoff: binding fold change ⁇ 0.75, SASA>10) and crystal structure (PDB: 2HEV) , which did not include the amino acids contributing to structure stability, such as amino acids belonging to CRD3 &CRD4. As shown in Table 15, there are thirteen hotspot positions to W3411-1.61.7-z19-p1-IgG1K.
  • a Fold change in binding is relative to the binding of several silent alanine substitutions.

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Abstract

La présente invention concerne des anticorps humanisés dirigés contre l'élément 4 de la superfamille des récepteurs du facteur de nécrose tumorale (TNFRSF4), également appelé OX40 et CD134. L'invention concerne également des procédés de génération d'hybridomes, les molécules d'acide nucléique codant pour les anticorps anti-OX40, les vecteurs d'expression et les cellules hôtes utilisés pour exprimer l'anticorps anti-OX40. L'invention concerne en outre des procédés pour valider la fonction des anticorps in vitro et l'efficacité des anticorps in vivo. Les anticorps de l'invention constituent un agent très puissant pour le traitement de multiples cancers par l'intermédiaire de la modulation de la fonction immunitaire humaine.
PCT/CN2019/125251 2018-12-14 2019-12-13 Anticorps humanisés dirigés contre ox40, procédé de préparation correspondant, et utilisation associée WO2020119793A1 (fr)

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Citations (5)

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CN102134276A (zh) * 2010-01-22 2011-07-27 上海抗体药物国家工程研究中心有限公司 一种抗ctla-4嵌合抗体
CN102216332A (zh) * 2008-10-31 2011-10-12 艾伯特生物治疗学公司 抗-cs1抗体用于治疗罕见淋巴瘤的用途
WO2013142300A2 (fr) * 2012-03-20 2013-09-26 Biogen Idec Ma Inc. Anticorps de neutralisation dirigés contre le jvc
WO2014148895A1 (fr) * 2013-03-18 2014-09-25 Biocerox Products B.V. Anticorps anti-cd134 (ox40) humanisés et leurs utilisations
CN108350053A (zh) * 2015-11-09 2018-07-31 英属哥伦比亚大学 淀粉样蛋白β表位及其抗体

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CN102216332A (zh) * 2008-10-31 2011-10-12 艾伯特生物治疗学公司 抗-cs1抗体用于治疗罕见淋巴瘤的用途
CN102134276A (zh) * 2010-01-22 2011-07-27 上海抗体药物国家工程研究中心有限公司 一种抗ctla-4嵌合抗体
WO2013142300A2 (fr) * 2012-03-20 2013-09-26 Biogen Idec Ma Inc. Anticorps de neutralisation dirigés contre le jvc
WO2014148895A1 (fr) * 2013-03-18 2014-09-25 Biocerox Products B.V. Anticorps anti-cd134 (ox40) humanisés et leurs utilisations
CN108350053A (zh) * 2015-11-09 2018-07-31 英属哥伦比亚大学 淀粉样蛋白β表位及其抗体

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