WO2017062615A2 - Combination therapy for the treatment of cancer - Google Patents

Combination therapy for the treatment of cancer Download PDF

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Publication number
WO2017062615A2
WO2017062615A2 PCT/US2016/055738 US2016055738W WO2017062615A2 WO 2017062615 A2 WO2017062615 A2 WO 2017062615A2 US 2016055738 W US2016055738 W US 2016055738W WO 2017062615 A2 WO2017062615 A2 WO 2017062615A2
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antibody
ctla
domain
cancer
administered
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PCT/US2016/055738
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French (fr)
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WO2017062615A3 (en
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James VASSELLI
Jon Marc WIGGINTON
Ezio Bonvini
Scott Koenig
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Macrogenics, Inc.
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Publication of WO2017062615A2 publication Critical patent/WO2017062615A2/en
Publication of WO2017062615A3 publication Critical patent/WO2017062615A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation.
  • the invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers.
  • the invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
  • B7 family members are immunoglobulin superfamily members with an immunoglobulin- V-like and an immunoglobulin-C-like domain (e.g., IgV-IgC) (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily;' Nature Rev. Immunol. 2: 116-126).
  • the IgV and IgC domains of B7-family members are each encoded by single exons, with additional exons encoding leader sequences, transmembrane and cytoplasmic domains.
  • the cytoplasmic domains are short, ranging in length from 19 to 62 amino acid residues and can be encoded by multiple exons (Collins, M. et al.
  • B7-1 CD80
  • B7-2 CD86
  • B7-1 (CD80) and B7-2 (CD86) exhibit dual specificity for the stimulatory CD28 receptor and the inhibitory CTLA-4 (CD 152) receptor (Sharpe, A.H. et al. (2002) “The B7-CD28 Superfamily;' Nature Rev. Immunol. 2: 116-126).
  • B7-H3 is unique in that the major human form contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC) (Collins, M. et al. (2005) "The B7 Family Of Immune-Regulatory Ligands," Genome Biol. 6:223.1-223.7). Although initially thought to comprise only 2 Ig domains (IgV-IgC) (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN- ⁇ Production;' Nature Immunol. 2:269-274; Sun, M. et al.
  • the 4Ig- B7-H3 molecule inhibits the natural killer cell-mediated lysis of cancer cells (Castriconi, R. et al. "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis," Proc. Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645).
  • the human B7-H3 (2Ig form) has been found to promote T-cell activation and IFN- ⁇ production by binding to a putative receptor on activated T cells (Chapoval, A.
  • B7-H3 A Costimulatory Molecule For T Cell Activation and IFN- ⁇ Production
  • Nature Immunol. 2:269-274 Xu, H. et al. (2009) "MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors " Cancer Res. 69(15):5275-6281).
  • Both B7-H4 and B7-H1 are potent inhibitors of immune function when expressed on tumor cells (Flies, D.B. et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity " J. Immunother. 30(3):251-260).
  • B7-H3 The mode of action of B7-H3 is complex, as the protein mediates both T cell co- stimulation and co-inhibition (Hofmeyer, K. et al. (2008) "The Contrasting Role OfB7-H3 " Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278; Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation And Anti-Tumor Immunity " Semin. Cancer Biol. 17(4):288-298; Subudhi, S.K. et al. (2005) "The Balance Of Immune Responses: Costimulation Verse Coinhibition " J. Mol. Med. 83 : 193-202).
  • B7-H3 binds to (TREM)-like transcript 2 (TLT-2) and co-stimulates T cell activation, but also binds to as yet unidentified receptor(s) to mediate co-inhibition of T cells.
  • B7-H3 through interactions with unknown receptor(s) is an inhibitor for natural killer cells and osteoblastic cells (Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278).
  • the inhibition may operate through interactions with members of the major signaling pathways through which T cell receptor (TCR) regulates gene transcription ⁇ e.g., NFTA, NF- ⁇ , or AP- 1 factors).
  • TCR T cell receptor
  • B7-H3 co-stimulates CD4+ and CD8+ T-cell proliferation.
  • B7-H3 also stimulates IFN- ⁇ production and CD8+ lytic activity (Chapoval, A. etal. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN- ⁇ Production " Nature Immunol. 2:269-274; Sharpe, A H. et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2: 116-126).
  • NFAT nuclear factor for activated T cells
  • NF-KB nuclear factor kappa B
  • AP-1 Activator Protein- 1
  • B7-H3 is also believed to inhibit Thl, Th2, or Thl7 in vivo (Prasad, D.V. etal. (2004) "Murine B7-H3 Is A Negative Regulator Of T Cells " J. Immunol. 173 :2500-2506; Fukushima, A.
  • Molecules that block the ability of a B7 molecule to bind to a T-cell receptor ⁇ e.g., CD28 inhibit the immune system and have been proposed as treatments for autoimmune disease (Linsley, P S. et al. (2009) “The Clinical Utility Of Inhibiting CD28-Mediated Co- Stimulation " Immunolog. Rev. 229:307-321).
  • Neuroblastoma cells expressing 4Ig-B7-H3 treated with anti-4Ig-B7-H3 antibodies were more susceptible to NK cells.
  • B7-H3 is not expressed on resting B or T cells, monocytes, or dendritic cells, but it is induced on dendritic cells by IFN- ⁇ and on monocytes by GM-CSF (Sharpe, A.H. et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2: 116-126).
  • the receptor(s) that bind B7-H3 have not been fully characterized. Early work suggested one such receptor would need to be rapidly and transiently up-regulated on T cells after activation (Loke, P. et al. (2004) "Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T Cells " Arthritis Res. Ther.
  • human B7-H3 In addition to its expression on neuroblastoma cells, human B7-H3 is over- expressed in a wide range of cancers and cultured cancer stem-like cells.
  • B7-H3 is broadly overexpressed on many malignant neoplasms including: SCCHN, where the level is directly proportional to the development of distal metastases and decreased survival (Katayama, A., et al. (2011) “Expression of B7-H3 In Hypopharyngeal Squamous Cell Carcinoma As A Predictive Indicator For Tumor Metastasis And Prognosis," Int J Oncol 38: 1219-26); bladder cancer (Boorjian, S.A., et al.
  • Cytotoxic T-lymphocyte associated protein-4 (CTLA-4; CD 152) is a single pass type I membrane protein that forms a disulfide linked homo-dimer (Schwartz J.C., et al. (2001) "Structural Basis For Co-Stimulation By The Human CTLA-4/B7-2 Complex " Nature 410:604-608).
  • Alternate splice variants, encoding different isoforms, have been characterized including a soluble isoform which functions as a monomer (Magistrelli G., et al.
  • CTLA-4 is primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalization (Alegre M-L, et al, (1996) "Regulation Of Surface And Intracellular Expression Of CTLA 4 On Mouse T Cells," J. Immunol. 157:4762-4770; Linsley, P.S., et al. (1996) "Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement,” Immunity 4:535-543).
  • CTLA- 4 is expressed at low levels on the surface of naive effector T cells (Alegre, M.L., et al. (1996) "Regulation Of Surface And Intracellular Expression Of CTLA4 On Mouse T Cells," J Immunol 157:4762-70), and constitutively expressed on T regulatory cells (Wang, X.B., et al.
  • the extracellular region of CTLA-4 comprises a single extracellular Ig(V) domain, followed by a transmembrane (TM) region and a small intracellular cytoplasmic tail (37 amino acids).
  • the intracellular tail contains two tyrosine-based motifs, which interact with several intracellular proteins, including the lipid kinase phosphatidylinositol 3 -kinase (PI3K), the phosphatases SHP-2 and PP2A and clathrin adaptor proteins AP-1 and AP-2 (Rudd, C.E. et al.
  • CTLA-4 is related to CD28, with the two proteins having approximately 29% identity at the amino acid level (Harper, K. (1991) "CTLA-4 And CD28 Activated Lymphocyte Molecules Are Closely Related In Mouse And Human As To Sequence, Message Expression, Gene Structure, And Chromosomal Location,” J. Immunol. 147: 1037-1044).
  • TCRs T cell receptors
  • CTLA-4 (CD 152) Can Inhibit T Cell Activation By Two Different Mechanisms Depending On Its Level Of Cell Surface Expression " J Immunol 165: 1352-6;Chuang, E., et al.
  • CTLA-4 acts as a negative regulator of T effector cell activation that diminishes effector function and dictates the efficacy and duration of a T cell response (Linsley, P.S., et al. (1996) "Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement ,” Immunity 4:535-43).
  • CTLA-4 may play a role in enhancing the negative effect of regulatory T cells on the immune response to cancer (Tai, Y.T., et al., (2012) "Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HM1.24 Antibody against Multiple Myeloma via Augmented Effector Function," Blood 119:2074-82).
  • CTLA-4 has a much higher affinity for members of the B7 family than for CD28, and therefore its expression on a T effector cell dictates a dominant negative regulation of the T cell (Allison, J.P., et al.
  • CTLA-4 contributes to the suppressor function of T regulatory cells.
  • the mechanism by which CTLA-4 contributes to the suppressor function of T regulatory cells is incompletely understood, but the expression of CTLA-4 on T regulatory cells enhances the suppressive function of these cells (Tai, Y.T., et al, (2012) "Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HMl .24 Antibody against Multiple Myeloma via Augmented Effector Function," Blood 119:2074-82).
  • B7-H3 Overexpression of B7-H3 occurs in a wide range of cancers and cultured cancer stem-like cells. As stated above, the overexpression of B7-H3 is strongly associated with increased disease recurrence and a poor prognosis. However, B7-H3 is a potential target for anti-B7-H3 drugs, including monoclonal antibodies that target the extracellular domain of the receptor. Antibodies and other molecules that specifically bind to B7-H3 have been described (see, United States Patent No. 8,802,091, 7,527,969; 7,368,554; 7,358,354; and 7,279,567; United States Patent Application Publications Nos.
  • the adaptive immune system can be a potent defense mechanism against cancer and disease, it is often hampered by immune suppressive mechanisms in the tumor microenvironment, such as the expression of co-inhibitory molecules.
  • co- inhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor milieu can dominantly attenuate T-cell responses against cancer cells.
  • novel combinations and treatment regimens that specifically recognize targets on the surface of cancer cells, and which can thereby mediate T-cell activation, simulation of an immune response and killing of cancer cells that express B7-H3. It is an object of this invention to identify such compositions and treatment regimens.
  • the present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation.
  • the invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers.
  • the invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
  • the invention provides a method of treating a cancer comprising administering to a subject in need thereof, a molecule that specifically binds to B7-H3, and a molecule that specifically binds to CTLA-4.
  • the invention particularly concerns embodiments of such methods, wherein the molecule that specifically binds to B7-H3 is an anti-B7-H3 antibody or antigen-binding fragment thereof, and the molecule that specifically binds to CTLA-4 is an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.
  • the invention particularly concerns the embodiment of such methods wherein the anti-B7-H3 antibody, or antigen-binding fragment thereof:
  • (b) has the three heavy chain CDRs and the three light chain CDRs of BRCA84D, hBRCA84D (1.1), hBRCA84D (2.2), hBRCA84D-2, hBRCA69D (1.1), hBRCA (2.2), or the three heavy chain CDRs and the three light chain CDRs of an anti-B7-H3 selected from Table 5; or
  • the invention particularly concerns the embodiment of such methods wherein the anti-CTLA-4 antibody, or antigen-binding fragment thereof:
  • the invention further concerns embodiments of such methods wherein the anti-B7- H3 antibody or antigen-binding fragment thereof comprises an Fc Domain, and/or the anti- CTLA-4 antibody, or antigen-binding fragment thereof comprises an Fc Domain.
  • the invention further concerns the embodiments of such methods wherein the anti-B7-H3 antibody or antigen-binding fragment thereof, comprises a variant Fc Domain having one, two, three, four, five or more substitutions in the Fc Domain that enhance(s) ADCC.
  • the invention further concerns the embodiments of such methods wherein the Fc Domain modifications that enhance ADCC comprise any one, any two, any three, any four, or all five of the substitutions L235V, F243L, R292P, Y300L and P396L, preferably of an IgGl Fc Domain.
  • the invention further concerns embodiments of such methods, wherein the anti-B7- H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is ipilimumab, or tremelimumab.
  • the invention further concerns embodiments of such methods, wherein the anti-B7-H3 antibody is hBRCA84D-2, and the anti-CTLA-4 antibody, or the antigen-binding fragment thereof, comprises the VH and VL Domains of ipilimumab, tremelimumab, or the VH and VL Domains of an anti-CTLA-4 antibody selected from Table 6.
  • the invention additionally concerns embodiments of such methods wherein the method comprises an Induction Period followed by a Maintenance Period, wherein:
  • the Induction Period comprises administering to a subject in need thereof a combination of an anti-B7-H3 antibody and an anti-CTLA-4 antibody, wherein
  • the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg recipient body weight weekly; and (ii) the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg recipient body weight every three weeks for a total of four doses of the anti-CTLA-4 antibody, and
  • (B) the Maintenance Period comprises:
  • the invention also concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage of 1-15 mg/kg recipient body weight and the molecule that specifically binds to CTLA- 4 (particularly an anti-CTLA-4 antibody) is administered at a dosage of 1-15 mg/kg recipient body weight.
  • the invention further concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage selected from 1 mg/kg, 3 mg/kg, 10 mg/kg and 15 mg/kg recipient body weight and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) is administered at a dosage selected from 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, and 15 mg/kg recipient body weight.
  • the molecule that specifically binds to B7-H3 is administered at a dosage selected from 1 mg/kg, 3 mg/kg, 10 mg/kg and 15 mg/kg recipient body weight
  • CTLA-4 particularly an anti-CTLA-4 antibody
  • the invention particularly concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage selected from 3 mg/kg, 10 mg/kg and 15 mg/kg recipient body weight, and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) is administered at a dosage of 3 mg/kg recipient body weight.
  • the invention further concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) are administered by IV infusion.
  • the invention also concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) are administered every three or twelve weeks within a 48 hour period.
  • the invention particularly concerns embodiments of such methods wherein the cancer is a cancer in which B7-H3 is expressed.
  • the cancer is a squamous cell cancer of the head and neck (SCCHN), a bladder cancer, a breast cancer, a colorectal cancer, a gastric cancer, a glioblastoma, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, a pharyngeal cancer, a prostate cancer, a renal cell carcinoma, a small round blue cell tumor, a neuroblastoma, or a rhabdomyosarcoma, in which B7-H3 is expressed.
  • SCCHN head and neck
  • the invention additionally concerns embodiments of such methods further comprising the step of administering a third therapeutic agent, particularly wherein the third therapeutic agent is an anti-angiogenic agent, an anti -neoplastic agent, a chemotherapeutic agent, or a cytotoxic agent
  • Figure 1 provides a schematic of a combination dosing schedule having an Induction Period followed by a Maintenance Period.
  • the present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation.
  • the invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers.
  • the invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
  • Antibodies are immunoglobulin molecules capable of immunospecific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., (an “antigen”) through at least one epitope recognition site, located in the Variable Domain of the immunoglobulin molecule.
  • the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, camelized antibodies, single-chain antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention), but also mutants thereof, naturally occurring variants, fusion proteins comprising an epitope binding site of the required immunospecificity, humanized antibodies, and chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required immunospecificity.
  • an "antigen-binding fragment" of an antibody is an immunoglobulin whose amino acid sequence comprises at least one epitope-binding site of an antibody specific for such antigen.
  • the term encompasses fragments (e.g., Fab, Fab', F(ab') 2 Fv), disulfide-linked bispecific Fvs (sdFv), intrabodies, and single-chain molecules (e.g., scFv).
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an epitope-binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • Natural antibodies are composed of two Light Chains complexed with two Heavy Chains.
  • Each Light Chain contains a Variable Light Chain (VL) Domain and a Constant Light Chain (CL) Domain.
  • Each heavy chain contains a Variable Heavy Chain (VH) Domain, three Heavy Chain Constant Domains (CHI, CH2 and CH3), and a Hinge Domain, which is located between the CHI and CH2 Domains.
  • VH Variable Heavy Chain
  • CHI Heavy Chain Constant Domain
  • CH2 and CH3 Three Heavy Chain Constant Domains
  • Hinge Domain which is located between the CHI and CH2 Domains.
  • the basic structural unit of naturally occurring immunoglobulins e.g., IgG
  • IgG immunoglobulins
  • each polypeptide chain includes its Variable (“V") Domain, which is of about 100 to 110 or more amino acids, and is primarily responsible for antigen recognition.
  • V Variable
  • C Constant
  • an intact, unmodified antibody e.g. , an IgG antibody
  • an immunoglobulin light and heavy chains i.e., the VL Domain and VH Domain, respectively.
  • Interaction of an antibody Light Chain and an antibody heavy chain and, in particular, interaction of its VL and VH Domains forms one of the epitope-binding sites of the antibody.
  • the Variable Domains of an IgG molecule consist of the complementarity determining regions (CDR), which contain the residues in contact with epitope, and non-CDR segments, referred to as framework segments (FR), which in general maintain the structure and determine the positioning of the CDR loops so as to permit such contacting (although certain framework residues may also contact antigen).
  • CDR complementarity determining regions
  • FR framework segments
  • the VL Domains have the structure (in the N-terminal to C-terminal direction): FR l 1-CDR L 1-FR L 2-CDR L 2-FR L 3-CDR L 3-FR l 4, and the VH Domains have the structure (in the N-terminal to C-terminal direction): FR h 1-CDRH1-FRH2-CDRH2-FR h 3- CDRH3-FRH4.
  • Polypeptides that are (or may serve as) the first, second and third CDR of an antibody Light Chain are herein respectively designated CDRLI Domain, CDRL2 Domain, and CDR L 3 Domain.
  • polypeptides that are (or may serve as) the first, second and third CDR of an antibody heavy chain are herein respectively designated CDRHI Domain, CDRH2 Domain, and CDRH3 Domain.
  • CDRLI Domain, CDRL2 Domain, CDRL3 Domain, CDRHI Domain, CDRH2 Domain, and CDRH3 Domain are directed to polypeptides that when incorporated into a protein cause that protein to be able to bind to a specific epitope regardless of whether such protein is an antibody having light and heavy chains or a single-chain binding molecule (e.g., an scFv, a BiTe, etc.), or is another type of protein.
  • the scFv construct comprises a VL and VH Domain of an antibody contained in a single polypeptide chain wherein the Domains are separated by a flexible linker of sufficient length to allow self-assembly of the two Domains into a functional epitope-binding site.
  • a linker of insufficient length less than about 12 amino acid residues
  • two of the scFv constructs may interact with one another other to form a bivalent molecule in which the VL of one chain associates with the VH of the other (reviewed in Marvin et al. (2005) "Recombinant Approaches To IgG-Like Bispecifw Antibodies, " Acta Pharmacol. Sin. 26:649-658).
  • antibodies In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. The last few decades have seen a revival of interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnology-derived drugs (Chan, C.E. et al. (2009) “The Use Of Antibodies In The Treatment Of Infectious Diseases," Singapore Med. J. 50(7):663-666). Nearly 200 antibody- based drugs have been approved for use or are under development. [0036] The term "monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non- naturally occurring) that are involved in the selective binding of an antigen.
  • Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site).
  • the term "monoclonal antibody” encompasses not only an intact monoclonal antibody and a full- length monoclonal antibody, but also a fragment thereof (such as an Fab, Fab', F(ab') 2 Fv), single-chain (scFv), a mutant thereof, a fusion protein comprising an antibody portion, a humanized monoclonal antibody, a chimeric monoclonal antibody, and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site of the required immunospecificity and the ability to bind to an epitope.
  • antibody it is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • the term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody.”
  • Methods of making monoclonal antibodies are known in the art. One method which may be employed is the method of Kohl er, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, ' " Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats or rabbits.
  • the antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that contain the desired epitope.
  • the immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, proteins, peptides, nucleic acids, or tissue.
  • Cells used for immunization may be cultured for a period of time (e.g. , at least 24 hours) prior to their use as an immunogen.
  • Cells may be used as immunogens by themselves or in combination with a non-denaturing adjuvant, such as Ribi (see, e.g., Jennings, V.M. (1995) "Review of Selected Adjuvants Used in Antibody Production," ILAR J. 37(3): 119- 125).
  • cells should be kept intact and preferably viable when used as immunogens. Intact cells may allow antigens to be better detected than ruptured cells by the immunized animal. Use of denaturing or harsh adjuvants, e.g., Freund's adjuvant, may rupture cells and therefore is discouraged.
  • the immunogen may be administered multiple times at periodic intervals such as, bi weekly, or weekly, or may be administered in such a way as to maintain viability in the animal (e.g., in a tissue recombinant).
  • existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for a desired pathogenic epitope can be sequenced and produced recombinantly by any means known in the art.
  • such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence of such antibodies may be used for genetic manipulation to generate a chimeric antibody, a humanized antibody, or a caninized antibody, or to improve the affinity, or other characteristics of the antibody.
  • humanized antibody refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and /or sequence of a human immunoglobulin.
  • the polynucleotide sequence of the variable domains of such antibodies may be used for genetic manipulation to generate such derivatives and to improve the affinity, or other characteristics of such antibodies.
  • the general principle in humanizing an antibody involves retaining the basic sequence of the antigen- binding portion of the antibody, while swapping the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody.
  • the epitope-binding site of such antibodies may comprise either complete Variable Domains fused onto Constant Domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the Variable Domains.
  • Antigen-binding sites may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224).
  • variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity determining regions
  • FRs framework regions
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which differ in sequence relative to the original antibody.
  • a number of "humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains (see, for example, Winter et al. (1991) "Man-made Antibodies " Nature 349:293-299; Lobuglio et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response " Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw et al.
  • CDRs complementarity determining regions
  • Natural antibodies are capable of binding to only one epitope species ⁇ i.e., they are "mono-specific"), although they may be able to bind multiple copies of that species ⁇ i.e., they may exhibit bi-valency or multi-valency).
  • the functionality of antibodies can be enhanced by generating multispecific antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating antibody- based molecule having higher valency ⁇ i.e., more than two binding sites) for the same epitope and/or antigen.
  • linker peptides either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further epitope-binding site ⁇ e.g.
  • WO 2013/174873, WO 2011/133886 and WO 2010/136172 teach tri- specific antibodies in which the CL and CHI Domains are switched from their respective natural positions and the VL and VH Domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind to more than one antigen.
  • PCT Publications Nos. WO 2013/163427 and WO 2013/119903 disclose modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain.
  • WO 2010/028797, WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc Domains have been replaced with additional VL and VH Domains, so as to form tri-valent binding molecules.
  • PCT Publications No. WO 2013/006544 discloses multi-valent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to yield heterodimeric structures.
  • WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose adding additional Binding Domains or functional groups to an antibody or an antibody portion (e.g., adding additional VL and VH Domains to the antibody's light and heavy chains, or adding a heterologous fusion protein or chaining multiple Fab Domains to one another).
  • Patent Publication 2007/ 0196363 concern bi-specific antibodies that are formed from the heavy chains of two antibodies, one of which possess a protuberance engineered into its heavy chain and the second of which possess a complementary cavity engineered into its heavy chain.
  • the presence of such complementary "knobs” and “holes” is taught to preferentially form bi- specific hetero-antibodies (having one heavy chain of each such antibody) relative to monospecific homo-antibodies that contain two heavy chains of the same antibody.
  • the CH2 and CH3 Domains of the two heavy chains interact to form the Fc Domain, which is a domain that is recognized by cellular Fc Receptors (FcyRs).
  • FcyRs Fc Receptors
  • the term "Fc Domain” is used to define a C-terminal region of an IgG heavy chain.
  • the amino acid sequence of the CH2-CH3 domain of an exemplary human IgGl is (SEQ ID NO:l):
  • amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG2 is (SEQ ID NO:2):
  • X is a lysine (K) or is absent.
  • amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG3 is (SEQ ID NO:3):
  • ALHNHYTQKS LSLSLGX as numbered by the EU index as set forth in Kabat, wherein, X is a lysine (K) or is absent.
  • the numbering of the residues in the constant region of an IgG heavy chain is that of the EU index as in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, H1, MD (1991), expressly incorporated herein by references.
  • the "EU index as in Kabat” refers to the numbering of the human IgGl EU antibody.
  • Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid.
  • Rabat's numbering scheme is extendible to antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids.
  • This method for assigning residue numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position to an amino acid at position 50 of a mouse antibody light chain.
  • the CH2 domain of a human IgG Fc Domain usually extends from amino acids 231 to amino acid 341 of a human IgG according to the EU numbering system of Kabat.
  • the CH3 domain of a human IgG usually extends from amino acids 342 to 447 according to the EU numbering system of Kabat.
  • the "hinge region” or “hinge domain” is generally defined as stretching from Glu216 to Pro230 of human IgGl .
  • Polymorphisms have been observed at a number of different positions within antibody constant regions ⁇ e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index as set forth in Kabat), and thus slight differences between the presented sequence and sequences in the prior art can exist. Polymorphic forms of human immunoglobulins have been well-characterized.
  • Gm Glm (1, 2, 3, 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5)
  • Glm 1, 2, 3, 17
  • Glm a, x, f, z
  • G2m G2m (23) or G2m (n)
  • G3m 5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28
  • G3m bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5)
  • Lefranc, et al The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78
  • the antibodies useful in the methods of the present invention may incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype or haplotype of the sequences provided herein.
  • the C-terminal amino acid residue (bolded above) of the CH3 Domain may be post-translationally removed.
  • the C-terminal residue of the CH3 Domain is an optional amino acid residue in the B7-H3-binding molecules and CTLA- 4-binding molecules provided herein.
  • binding molecules lacking the C-terminal residue of the CH3 Domain.
  • binding molecules comprising the C-terminal lysine residue of the CH3 Domain are also encompassed.
  • the CHI Domain and/or hinge region may be of any isotype (e.g., IgGl, IgG2, IgG3, or IgG4), but is preferably of the same isotype as the desired Fc Domain.
  • amino acid sequence of an exemplary human IgGl CHI Domain is (SEQ ID NO:8):
  • amino acid sequence of an exemplary human IgG2 CHI Domain is (SEQ ID NO:9):
  • amino acid sequence of an exemplary human IgG4 CHI Domain is (SEQ ID NO:10):
  • amino acid sequence of an exemplary human IgGl hinge region is (SEQ ID NO: ll): EPKSCDKTHTCPPCP
  • amino acid sequence of an exemplary human IgG2 hinge region is (SEQ ID NO: 12):
  • amino acid sequence of an exemplary human IgG4 hinge region is (SEQ ID NO: 13): ESKYGPPCPSCP
  • Activating and inhibitory signals are transduced through the Fc gamma Receptors (FcyRs) following their ligation to an Fc Domain.
  • FcyRs Fc gamma Receptors
  • ITAMs immunoreceptor tyrosine- based activation motifs
  • ITFMS immunoreceptor tyrosine-based inhibitory motifs
  • ITAM-containing FcyR complexes include FcyRI, FcyRIIA, FcyRIIIA, whereas ITIM-containing complexes only include FcyRIIB.
  • Human neutrophils express the FcyRIIA gene.
  • FcyRIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation.
  • ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates (e.g., PbK). Cellular activation leads to release of proinflammatory mediators.
  • the FcyRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds IgG complexes in an indistinguishable manner.
  • the presence of an ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently the molecular basis of this inhibition was established.
  • the ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of the inositol polyphosphate 5 '-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of ITAM-containing FcyR-mediated tyrosine kinase activation, consequently preventing the influx of intracellular Ca ++ .
  • SHIP inositol polyphosphate 5 '-phosphatase
  • cross-linking of FcyRIIB dampens the activating response to FcyR ligation and inhibits cellular responsiveness. B cell activation, B cell proliferation and antibody secretion is thus aborted.
  • interaction with the neonatal Fc Receptor (FcRn) mediates the recycling of IgG molecules from the endosome to the cell surface and release into the blood.
  • Modification of the Fc Domain normally leads to an altered phenotype, for example altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function. It may be desirable to modify the Fc Domain of the B7-H3 -binding molecules and/or CTLA-4-binding molecules (e.g., anti-B7-H3 and anti-CTLA-4 antibodies) for use in the methods of the present invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies whose mechanism of action involves blocking or antagonism, but not killing of the cells bearing a target antigen.
  • CTLA-4-binding molecules e.g., anti-B7-H3 and anti-CTLA-4 antibodies
  • Increased effector function is generally desirable when directed to undesirable cells, such as tumor and foreign cells, where the FcyRs are expressed at low levels, for example, tumor-specific B cells with low levels of FcyRIIB (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma).
  • FcyRIIB e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma
  • the Fc Domain of the B7-H3-binding molecules and/or CTLA-4-binding molecules for use in the methods of the present invention may be either a complete Fc Domain (e.g., a complete IgG Fc Domain) or only a fragment of a complete Fc Domain.
  • the Fc Domain of the molecules useful in the methods of the present invention that contain such a domain may include some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete Fc Domain, or may comprise a variant CH2 and/or a variant CH3 sequence (that may include, for example, one or more insertions and/or one or more deletions with respect to the CH2 or CH3 domains of a complete Fc Domain).
  • Such Fc Domains may comprise non-Fc polypeptide portions, or may comprise portions of non-naturally complete Fc Domains, or may comprise non-naturally occurring orientations of CH2 and/or CH3 domains (such as, for example, two CH2 domains or two CH3 Domains, or in the N-terminal to C-terminal direction, a CH3 Domain linked to a CH2 Domain, etc.).
  • the Fc Domain of the B7-H3-binding molecules and/or CTLA-4-binding molecules may possess the ability to bind to one or more Fc receptors (e.g., FcyR(s)), more preferably such Fc Domain will cause altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by a wild-type Fc Domain) or will substantially eliminate the ability of such Fc Domain to bind to one or more FcyR (e.g., inhibitory receptor(s)).
  • FcyR(s) FcyR(s)
  • the molecules for use in the methods of the present invention may comprise a variant Fc Domain having altered affinities for an activating and/or inhibitory Fey receptor.
  • the molecules comprise a variant Fc Domain that has increased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain.
  • the molecules for use in the methods of the present invention comprise a variant Fc Domain that has decreased affinity for FcyRIIB and increased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain.
  • molecules for use in the methods of the present invention comprise a variant Fc Domain that has decreased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain.
  • the molecules for use in the methods of the present invention comprise a variant Fc Domain that has unchanged affinity for FcyRIIB and decreased (or increased) affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain.
  • the molecules for use in the methods of the present invention comprise a variant Fc Domain having an altered affinity for FcyRIIIA and/or FcyRIIA such that the immunoglobulin has an enhanced effector function.
  • effector cell functions include antibody dependent cell mediated cytotoxicity, antibody dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, Clq binding, and complement dependent cell mediated cytotoxicity.
  • Variant Fc Domains are well known in the art, and any known variant Fc Domain may be used in the present invention to confer or modify the effector function exhibited by a molecule comprising an Fc Domain (or portion thereof) as functionally assayed, e.g., in an NK dependent or macrophage dependent assay.
  • Fc Domain variants identified as altering effector function are disclosed in PCT Publications No. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; and WO 2008/140603, and any suitable variant disclosed therein may be used in the present molecules.
  • Table 4 lists exemplary single, double, triple, quadruple and quintuple Fc Domain
  • Still more particularly preferred variants include one or more modifications selected from Groups 1-105:
  • the invention encompasses B7-H3-binding molecules that comprise a variant Fc Domain wherein the variant confers or has an increased ADCC activity and/or an increased binding to FcyRIIIA (CD16A), and may also have a reduced binding to FcyRIIB (CD32B).
  • Exemplary variants of human IgGl Fc Domains with increased binding to CD16A and which may additionally have reduced binding to CD32B contain L235V, F243L, R292P, Y300L, V305I or P296L substitutions.
  • a B7-H3-binding molecule will comprise a variant Fc Domain having at least one modification in the Fc Domain.
  • the variant Fc Domain comprises at least one substitution selected from the group consisting of L235V, F243L, R292P Y300L, V305L and P396L.
  • the variant Fc Domain comprises:
  • the variant Fc Domain comprises substitutions of:
  • the CTLA-4-binding molecule comprises a variant Fc Domain wherein the variant confers or has decreased (or substantially no) binding to FcyRIIIA (CD 16a), relative to the binding exhibited by the wild-type Fc Domain (SEQ ID NO:l)).
  • Exemplary variants of human IgGl Fc Domains with reduced binding to FcyRs contain L234A, L235A, D265A, N297A or N297Q, substitutions.
  • Alternative CTLA-4- binding molecules useful in the invention include variant IgGl Fc Domains that include any 1, 2, 3, 4, or all 5, of the substitutions: L234A, L235A, D265A, N297A, and N297Q. These amino acid substitutions may be present in a human IgGl Fc Domain in any combination.
  • a CTLA-4-binding molecule will comprise a variant Fc Domain having at least one modification in the Fc Domain.
  • the variant Fc Domain comprises at least one substitution selected from the group consisting of L234A, L235A, D265A, and N297Q. Since the L234A, L235A, D265A, N297A, and N297Q substitutions abolish effector function, in circumstances in which effector function is desired, these substitutions would preferably not be employed.
  • the variant Fc Domain comprises substitutions of:
  • a preferred IgGl sequence for the CH2 and CH3 Domains of a variant Fc Domain with reduced binding to FcyRs for use in the methods of the invention will have the
  • the CH2-CH3 Domain of an Fc Domain-containing CTLA-4-binding molecule for use in the methods of the present invention may be one that inherently exhibits decreased (or substantially no) binding to FcyRIIIA (CD 16a) and/or reduced effector function (relative to the binding exhibited by the wild-type IgGl Fc Domain (SEQ ID NO:l)).
  • the CH2-CH3 Domain of an Fc Domain-containing CTLA-4- binding molecule for use in the methods of the present invention may be an IgG2 Fc Domain or an IgG4 Fc Domain.
  • a CTLA-4-binding molecule for use in the methods of the present invention comprises an IgG4 Fc Domain.
  • an IgG4 Fc Domain is utilized the instant invention also encompasses the introduction of a stabilizing hinge mutation such as S228P (e.g., ESKYGPPCPPCP (SEQ ID NO:14)), as numbered by the EU index as set forth in Kabat (Lu et al, (2008) "The Effect Of A Point Mutation On The Stability Of IgG4 As Monitored By Analytical Ultracentrifugation,” J. Pharm. Sci. 97:960-969) to reduce the incidence of strand exchange.
  • S228P e.g., ESKYGPPCPPCP (SEQ ID NO:14)
  • antibody will comprise an IgG4 CHI (see, e.g., SEQ ID NO: 10), a stabilized IgG4 hinge (see, e.g., SEQ ID NO:47), and IgG4 CH2-CH3 Domains (see, e.g., SEQ ID NO:4),
  • the serum half-life of proteins comprising Fc Domains may be increased by increasing the binding affinity of the Fc Domain for FcRn.
  • half-life as used herein means a pharmacokinetic property of a molecule that is a measure of the mean survival time of the molecules following their administration.
  • Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known quantity of the molecule from the subject's body (e.g., human patient or other mammal) or from a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues.
  • an increase in half-life for an administered molecule results in an increase in that molecule's mean residence time (MRT) in circulation.
  • MRT mean residence time
  • the B7-H3-binding molecules and/or CTLA-4-binding molecules for use in the methods of the present invention comprise a variant Fc Domain, wherein the variant Fc Domain comprises at least one amino acid modification relative to a wild-type Fc Domain, such that the molecule has an increased half-life (relative to that of a wild-type Fc Domain).
  • the B7-H3-binding molecules and/or CTLA-4-binding molecules for use in the methods of the present invention comprise a variant Fc Domain, wherein the variant Fc Domain comprises a half-live extending amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436.
  • the variant Fc Domain comprises a half-live extending amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 4
  • Fc Domain-containing molecules with enhanced half-life also include those with substitutions at two or more of Fc Domain residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435 and 436.
  • two or more substitutions selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, Y436I.
  • the variant Fc Domain comprises substitutions of:
  • the instant invention further encompasses variant Fc Domains comprising:
  • the two CH2 and/or two CH3 Domains of the CH2-CH3 Domains of two interacting Fc Domain-containing polypeptide chains of an B7-H3-binding molecules and/or CTLA-4-binding molecule for use in the methods of the present invention need not be identical in sequence, and advantageously are modified to foster complexing between the two polypeptide chains (see, for example: WO 98/50431; WO2007/110205; WO2011/143545; WO 2012/058768; WO 2013/06867).
  • an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a "knob", e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., "the hole” (e.g., a substitution with glycine).
  • the hole e.g., a substitution with glycine
  • the "knob” is engineered into the CH2-CH3 Domains of one polypeptide chain and the "hole” is engineered into the CH2-CH3 Domains of the other CH2- CH3 -containing polypeptide chain.
  • the "knob” will help in preventing the first polypeptide chain from homodimerizing via its CH2 and/or CH3 Domains.
  • the CH2-CH3 "hole-bearing" polypeptide chain will heterodimerize with the CH2-CH3 "knob-bearing” polypeptide chain, and will also homodimerize with itself.
  • a preferred knob is created by modifying a native IgG Fc Domain to contain the modification T366W.
  • a preferred hole is created by modifying a native IgG Fc Domain to contain the modification T366S, L368A and Y407V.
  • the protein A binding site of the CH2 and CH3 Domains of the "hole-bearing" Fc Domain is preferably mutated by amino acid substitution at position 435 (H435R).
  • the "hole-bearing" Fc Domain homodimer will not bind to protein A, whereas an Fc Domain-containing B7-H3-binding molecule and/or CTLA-4-binding molecule for use in the methods of the present invention will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.
  • a preferred "knob-bearing" sequence for an Fc Domain-containing B7-H3- binding molecule and/or CTLA-4-binding molecule has the sequence (SEQ ID NO:6):
  • a preferred "hole-bearing" sequence for an Fc Domain-containing B7-H3-binding molecule and/or CTLA-4-binding molecule has the sequence (SEQ ID NO:7):
  • the invention also encompasses such CH2-CH3 Domains, which comprise additional substitutions which modify effector function and/or FyR binding activity of the Fc Domain as provided above.
  • the invention also encompasses such CH2-CH3 Domains, which further comprise one or more half-live extending amino acid substitutions.
  • the invention encompasses such hole-bearing and such knob-bearing CH2-CH3 Domains which further comprise the M252Y/S254T/T256E substitutions.
  • Molecules that specifically bind B7-H3 encompassed by the invention include anti- B7-H3 antibodies capable of binding to a continuous or discontinuous (e.g., conformational) portion (epitope) of human B7-H3, and molecules comprising the epitope-binding site of such antibodies.
  • the B7-H3-binding molecules used in the methods and compositions of the present invention will preferably also exhibit the ability to bind to the B7-H3 molecules of one or more non-human species, especially, murine, rodent, canine, and primate species.
  • Antibodies that are specific for B7-H3 are known (see, e.g., United States Patent Nos.
  • Human B7-H3 exists as a "2Ig” form and as a "4Ig” form.
  • the amino acid sequence of the "2Ig” form of human B7-H3 (including a 29 amino acid residue signal sequence, shown underlined) is (SEQ ID NO: 18):
  • Preferred anti-B7-H3-binding molecules possess the VL and/or VH Domains, of the anti-human B7-H3 monoclonal antibody "BRCA84D,” “BRCA69D,” “PRCA1,” or any of the anti-B7-H3 antibodies provided in Table 5; and more preferably possess 1, 2 or all 3 of the CDRLS of the VL Region and/or 1, 2 or all 3 of the CDRHS of the VH Domain of such anti- B7-H3 monoclonal antibodies.
  • B7-H3-binding molecules which possess a humanized VH and/or VL Domain.
  • Such preferred B7-H3-binding molecules include antibodies having variant Fc Domains, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, etc.
  • any antibody comprising one of the humanized VL Domains paired with the humanized VH Domain is referred to generically as "hBRCA84D," and particular combinations of humanized VH/VL Domains are referred to by reference to the specific VH/VL Domains, for example a humanized antibody comprising hBRCA84D VHl and hBRCA84D VL2 is specifically referred to as “hBRCA84D (1.2)."
  • hBRCA69D VL1 Two exemplary humanized VL Domains of BRCA69D designated herein as "hBRCA69D VL1," and “hBRCA69D VL2,” and two exemplary humanized VH Domains of BRCA69D designated herein as “hBRCA69D VHl,” and “hBRCA69D VH2,” are provided below.
  • hBRCA69D VL2 includes amino acid substitutions in CDRLI and CDRL2
  • hBRCA69D VH2 includes amino acid substitutions in CDRL2.
  • Any of the humanized VL Domains may be paired with any of the humanized VH Domains to generate a B7-H3 binding domain.
  • any antibody comprising one of the humanized VL Domains paired with the humanized VH Domain is referred to genetically as "hBRCA69D," and particular combinations of humanized VH/VL Domains are referred to by reference to the specific VH/VL Domains, for example a humanized antibody comprising hBRCA69D VHl and hBRCA69D VL2 is specifically referred to as “hBRCA69D (1.2)."
  • B7-H3 antibodies useful in the methods and compositions of the instant inventions comprise the VL and VH Domains of any of the antibodies provided above (e.g., hBRCA84D, hBRCA69D, PRCA157, or the VL and VH Domains of any of the anti-B7-H3 antibodies in Table 5), a kappa CL Domain, and a variant IgGl Fc Domain having enhanced ADCC (relative to a wild-type Fc Domain).
  • the CH2-CH3 Domains comprise the L235V, F243L, R292P, Y300L and P396L substitutions (wherein the numbering is according to the EU index as in Kabat).
  • Such antibodies will preferably comprise an IgGl CHI Domain and Hinge Domain.
  • R292P, Y300L and P396L substitutions (SEQ ID NO:17) is shown below.
  • An exemplary anti-B7-H3 antibody designated "hBRCA84D-2,” comprises: a light chain having the VL Domain of BRCA84D VL2 (SEQ ID NO:23) and a kappa CL (SEQ ID NO: 15); and a heavy chain having the VH Domain of BRCA84D VH2 (SEQ ID NO:29), an IgGl CHI Domain and Hinge (SEQ ID NO: 16), and variant IgG CH2-CH3 Domains comprising L235V, F243L, R292P, Y300L and P396L substitutions (SEQ ID NO: 17).
  • Molecules that specifically bind CTLA-4 encompassed by the invention include anti-CTLA-4 antibodies capable of binding to a continuous or discontinuous (e.g., conformational) portion (epitope) of human CTLA-4, and molecules comprising the epitope- binding site of such antibodies.
  • the CTLA-4-binding molecules used in the methods and compositions of the present invention will preferably also exhibit the ability to bind to the CTLA-4 molecules of one or more non-human species, especially, murine, rodent, canine, and primate species.
  • Antibodies that are specific for CTLA-4 are known (see, e.g., United States Patents 6,984,720; 6,682,736; 7,034,121; 7,109,003; 7,132,281; 7,411,057; 7,605,238; 7,807,797; 7,824,679; 8,017,114; 8,143,379; 8,318,916; 8,491,895; 8,784,815; 8,883,984; US Patent Publications 2009/0123477; 2009/0252741; 2014/0105914; and PCT Patent Publications WO 00/37504; WO 01/14424; WO 01/54732; WO 2006/029219; WO 2006/066568; WO 2012/120125; and Table 6). Additional desired antibodies may be made by isolating antibody-secreting hybridomas elicited using CTLA-4 or a peptide fragment thereof.
  • Human CTLA-4 (including a 35 amino acid residue signal sequence (shown underlined) and the 188 amino acid residue mature protein) has the amino acid sequence (SEQ ID NO:42):
  • Preferred anti-CTLA-4-binding molecules useful in the methods and compositions of the instant invention possess the VL and/or VH Domains of the anti-human CTLA-4 monoclonal antibody "CTLA-4 mAb 1" (ipilimumab, CAS Reg. No.:477202-00-9, also known as MDX010, and marketed as Yervoy® by Bristol-Myers Squibb); "CTLA-4 mAb 2" (tremelimumab, CAS Reg.
  • VL and/or VH Domains of any of the anti-CTLA-4 antibodies in Table 6 and more preferably possess 1, 2 or all 3 of the CDRLS of the VL Region and/or 1, 2 or all 3 of the CDRHS of the VH Domain of such anti-CTLA-4 monoclonal antibodies.
  • preferred anti-CTLA-4-binding molecules include antibodies having wild-type Fc Domains, variant Fc Domains, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, etc.
  • CTLA-4 antibodies useful in the methods and compositions of the instant inventions comprise the VL and VH Domains of any of the antibodies provided above (e.g., CTLA-4 mAb 1, CTLA-4 mAb 2 or any of the anti-CTLA-4 antibodies in Table 6), a kappa CL Domain, and an IgGl, IgG2 or IgG4 Fc Domain, optionally lacking the C- terminal lysine residue.
  • Such antibodies will preferably comprise a CHI Domain and a Hinge Domain of the same isotype as the Fc Domain.
  • An exemplary anti-CTLA antibody is ipilimumab (CAS Reg. No.:477202-00-9), which is a human antibody comprising a light chain having the VL Domain of CTLA-4 mAb 1 (SEQ ID NO:44), and a kappa CL Domain (see, e.g., SEQ ID NO:15); and a heavy chain have the VH Domain of CTLA-4 mAb 1 (SEQ ID NO:43), an IgGl CHI Domain (see, e.g., SEQ ID NO:8), an IgG Hinge (see, e.g., SEQ ID NO:ll), and IgGl CH2-CH3 Domains (see, e.g., SEQ ID NO:l)
  • Another exemplary anti-CTLA antibody is tremelimumab (CAS Reg. No. : 745013- 59-6), which is a human antibody comprising a light chain having the VL Domain of CTLA-4 mAb 2 (SEQ ID NO:46), and a kappa CL Domain (see, e.g., SEQ ID NO:15); and a heavy chain having the VH Domain of CTLA-4 mAb 12 (SEQ ID NO: 45), an IgG2 CHI Domain (see, e.g., SEQ ID NO:9), an IgG2 Hinge (see, e.g., SEQ ID NO:12), and IgG2 CH2-CH3 Domains (see, e.g., SEQ ID NO:2).
  • B7-H3-binding molecules and CTLA-4-binding molecules encompassed by the present invention can be produced by methods known in the art, for example, synthetically or recombinantly (see, e.g., Kelley, R. F. et al. (1990) In: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, J.K. Ed., Plenum Press, N.Y., vol. 12, pp 1-19; Stewart, J.M et al. (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL; see also United States Patents Nos.
  • suitable B7-H3-binding molecules and/or CTLA-4-binding molecules having one or more of the CDRs of a desired anti-B7-H3 antibody and/or anti- CTLA-4 antibody may be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XENOMOUSETM (Abgenix, Inc., Fremont, CA) and HUMAB -MOUSE® and TC MOUSETM (both from Medarex, Inc., Princeton, NJ).
  • binding molecules may be made recombinantly and expressed using any method known in the art.
  • Antibodies may be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that may be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been disclosed (see, for example, Peeters et al. (2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19:2756; Lonberg, N. et al.
  • Vectors containing polynucleotides encoding polypeptides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
  • infection e.g., where the vector is an infectious agent such as vaccinia virus.
  • the choice of introducing vectors or polynucleotides will often depend on features of the host cell.
  • Any host cell capable of overexpressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
  • suitable mammalian host cells include but are not limited to COS, HeLa, and CHO cells.
  • the host cells express the cDNAs at a level of about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells.
  • Screening the host cells for immunospecific binding to a cDNA expressed target may be accomplished using an immunoassay or FACS.
  • a cell overexpressing the antibody or protein of interest can be identified.
  • the invention includes polypeptides comprising an amino acid sequence (preferably the epitope binding domain) of an anti-B7-H3 antibody and/or anti-CTLA-4- antibody provided herein.
  • the polypeptides of this invention can be made by procedures known in the art.
  • the polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis. Polypeptides, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis.
  • the invention includes modifications of the polypeptides of any such B7-H3- binding molecules and/or CTLA-4-binding molecules that do not significantly affect the properties of such molecules as well as variants that have enhanced or decreased activity.
  • the modification of polypeptides is a routine practice in the art and need not be described in detail herein.
  • Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.
  • Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine.
  • These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation.
  • the amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar chemical properties as that of the original amino acid.
  • conservative substitutions are known in the art, and examples have been provided above.
  • Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the Variable Domain. Changes in the Variable Domain can alter binding affinity and/or immunospecificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as the attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art.
  • the invention encompasses fusion proteins comprising one or more of the antibodies of this invention.
  • a fusion polypeptide is provided that comprises a light chain, a heavy chain or both a light and heavy chain.
  • the fusion polypeptide contains a heterologous immunoglobulin constant region.
  • the fusion polypeptide contains a VL Domain and a VH Domain of an antibody provided herein or produced from a publicly-deposited hybridoma.
  • an antibody fusion protein contains one or more epitope-binding sites that immunospecifically bind to B7-H3 and/or CTLA-4, and one or more polypeptide domains that immunospecifically bind to another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region.
  • compositions comprising a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules.
  • the compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms.
  • Such compositions comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules, and a pharmaceutically acceptable carrier.
  • such compositions are substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side effects).
  • the agents may be formulated together in the same formulation or may be formulated into separate compositions. Accordingly, in some embodiments, the B7-H3 -binding molecule and the CTLA-4-binding molecule are formulated together in the same pharmaceutical composition. In alternative embodiments, the molecules are formulated in separate pharmaceutical compositions.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that are well known in the art and are relatively inert substances that facilitate administration of a pharmacologically effective substance or which facilitate processing of the active compounds into preparations that can be used pharmaceutically for delivery to the site of action.
  • excipients can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, buffers, and skin penetration enhancers.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
  • adjuvant e.g., Freund's adjuvant (complete and incomplete)
  • excipient e.g., complete and incomplete
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Aqueous carriers such as saline solutions, aqueous dextrose and glycerol solutions are preferred when the pharmaceutical composition is administered intravenously.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain a minor amount of a wetting or emulsifying agent, or a pH buffering agent. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided, so that the ingredients may be mixed prior to administration.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, but are not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the therapeutic agent ⁇ i.e., a B7-H3-binding molecule, a CTLA-4- binding molecule, or a combination of such molecules
  • a dry sterile lyophilized powder in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule(s).
  • the therapeutic agent is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.
  • the therapeutic agent is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the therapeutic agent.
  • the lyophilized therapeutic agent ⁇ i.e., a B7-H3-binding molecule, a CTLA-4- binding molecule, or a combination of such molecules
  • the agent should be stored at between 2°C and 8°C in the original container and the agent should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • the therapeutic agent is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule(s), fusion protein, or conjugated molecule.
  • such therapeutic agent when provided in liquid form is supplied in a hermetically sealed container.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers containing a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules alone or with other agents, preferably with a pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • a kit can comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules.
  • the kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens.
  • the other prophylactic or therapeutic agent is a chemotherapeutic agent.
  • the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.
  • the present invention provides methods of treating cancer, comprising administering to a subject in need thereof a B7-H3-binding molecule and a CTLA- 4-binding molecule.
  • the present invention encompasses such methods wherein, the B7-H3-binding molecule comprises the epitope-binding site of an anti-B7-H3 antibody provided herein, and wherein the CTLA-4-binding molecule comprises the epitope-binding site of an anti-CTLA-4 antibody provided herein.
  • the B7-H3-binding molecule is an antibody.
  • the CTLA-4-binding molecule is an antibody.
  • both the B7-H3 -binding molecule and CTLA-4-binding molecule are antibodies.
  • a B7-H3-binding molecule and a CTLA-4-binding molecule are administered concurrently.
  • concurrent administration is intended to denote:
  • A the administration of a single pharmaceutical composition that contains both a B7-H3-binding molecule and a CTLA-4-binding molecule.
  • Such molecules may be the same molecule (e.g., a bispecific antibody), or may be distinct (e.g., an anti-B7-H3 antibody, or antigen-binding fragment thereof, and an anti- CTLA-4-antibody, or antigen-binding fragment thereof); or
  • compositions (B) the separate administration of two or more pharmaceutical compositions, one composition of which contains a molecule that specifically binds to B7-H3 and another composition of which contains a molecule that specifically binds to CTLA-4, wherein the compositions are administered within a 48-hour period.
  • two distinct molecules are employed, and the molecules are administered "sequentially" (e.g., an anti-B7-H3 antibody is administered and, at a later time, an anti-CTLA-4 antibody is provided, or vice versa).
  • the second administered composition is administered at least 48 hours, or more after the administration of the first administered composition.
  • Providing a therapy or "treating” refers to any indicia of beneficial or desired results including, without limitation, clinical results such as shrinking the size of a tumor (in the cancer context, for example, a tumor of breast, gastric or prostate cancer), retardation of cancer cell growth, delaying the development of metastasis, decreasing a symptom resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and/or prolonging survival of an individual.
  • clinical results such as shrinking the size of a tumor (in the cancer context, for example, a tumor of breast, gastric or prostate cancer), retardation of cancer cell growth, delaying the development of metastasis, decreasing a symptom resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying
  • Subjects for treatment include animals, most preferably mammalian species such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as a cynomolgus monkey, human, etc.).
  • a non-primate e.g., bovine, equine, feline, canine, rodent, etc.
  • a primate e.g., monkey such as a cynomolgus monkey, human, etc.
  • the subject is a human.
  • Exemplary disorders that may be treated by various embodiments of the present invention include, but are not limited to, proliferative disorders, cell proliferative disorders, and cancer (especially a B7-H3 -expressing cancer).
  • the invention encompasses methods and compositions for treatment, prevention or management of a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount a molecule that specifically binds to B7-H3 and a molecule that specifically binds to CTLA-4.
  • the B7-H3-binding molecule and the CTLA-4-binding molecule are particularly useful for the prevention, inhibition, reduction of growth or regression of primary tumors, and metastasis of cancer cells.
  • binding molecules may mediate effector function against cancer cells, promote the activation of the immune system against cancer cells, cross-link cell-surface antigens and/or receptors on cancer cells and enhance apoptosis or negative growth regulatory signaling, or a combination thereof, resulting in tumor clearance and/or tumor reduction.
  • an "effective amount" of a pharmaceutical composition in one embodiment, is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as decreasing symptoms resulting from the disease attenuating a symptom of infection (e.g., viral load, fever, pain, sepsis, etc.) or a symptom of cancer (e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.), thereby increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and/or prolonging survival of individuals.
  • a symptom of infection e.g., viral load, fever, pain, sepsis, etc.
  • a symptom of cancer e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.
  • a B7-H3-binding molecule e.g., an antibody
  • a CTLA-4- binding molecule e.g., an antibody
  • the methods and compositions of the instant invention may be used for immunotherapy directed at cancer including cancers characterized by the presence of a cancer cell, including but not limited to a cell of an acute myeloid leukemia, an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dys
  • the combination of a B7-H3-binding molecule and a CTLA-4-binding molecule is particularly useful for the treatment of squamous cell cancers of the head and neck (SCCHN), bladder cancers, breast cancers, colorectal cancers, gastric cancers, glioblastomas, kidney cancers, lung cancers including non-small cell lung cancers (NSCLC), melanomas, ovarian cancers, pancreatic cancers, pharyngeal cancers, prostate cancers, renal cell carcinomas, and small round blue cell tumors of childhood including neuroblastomas and rhabdomyosarcomas, each of which highly express B7-H3.
  • SCCHN head and neck
  • NSCLC non-small cell lung cancers
  • melanomas melanomas
  • ovarian cancers pancreatic cancers
  • pharyngeal cancers prostate cancers
  • renal cell carcinomas small round blue cell tumors of childhood including neuroblastomas and rhabdomyosarcom
  • the B7-H3-binding molecules and CTLA-4-binding molecules are administered at a concentration that promotes binding at physiological (e.g., in vivo) conditions.
  • the B7-H3-binding molecules and CTLA-4-binding molecules e.g., anti-B7-H3 and anti-CTLA-4 antibodies
  • additional agents that enhance or direct an individual's own immune response such as an agent that strengthens ADCC or stimulated T-cells.
  • the B7-H3-binding molecule and/or the CTLA-4- binding molecule may be conjugated to or associated with a radioactive molecule, toxin (e.g., calicheamicin), chemotherapeutic molecule, liposomes or other vesicles containing chemotherapeutic compounds and administered to an individual in need of such treatment to target these compounds to the cancer cell containing the antigen recognized by the B7-H3- binding molecule and thus eliminate cancer or diseased cells.
  • a radioactive molecule e.g., toxin (e.g., calicheamicin)
  • chemotherapeutic molecule e.g., liposomes or other vesicles containing chemotherapeutic compounds
  • the B7-H3-binding molecule e.g., an anti-B7-H3 antibody
  • the B7-H3-binding molecule is internalized by the cell bearing B7-H3 at its surface, thus delivering the conjugated moiety to the cell to induce the therapeutic effect and the CTLA-4-binding molecule promotes the activation of the immune system.
  • the B7-H3-binding molecule and CTLA-4-binding molecule can be employed as an adjuvant therapy at the time of the surgical removal of a tumor in order to delay, suppress or prevent the development of metastasis.
  • the molecules can also be administered before surgery (neoadjuvant therapy) in order to decrease the size of the tumor and thus enable or simplify surgery, spare tissue during surgery, and/or decrease any resulting disfigurement.
  • Molecules having an Fc Domain with an increased affinity for FcyRIIIA and/or FcyRIIA, and optionally a decreased affinity for FcyRIIB may lead to an enhanced activating response upon FcyR binding and thus have enhanced therapeutic efficacy for treating and/or preventing cancer.
  • B7-H3-binding molecules comprising a variant Fc Domain are particularly useful for the treatment and/or prevention of a disease, or disorder where an effector cell function (e.g., ADCC) mediated by FcyR is desired (e.g., cancer).
  • a B7-H3 -binding molecule having enhanced FcyRIIIA binding may bind a cell-surface antigen and FcyRIIIA on an immune effector cell (e.g., K cell), stimulating an effector function (e.g., ADCC, CDC, phagocytosis, opsonization, etc.) against the cell.
  • an immune effector cell e.g., K cell
  • stimulating an effector function e.g., ADCC, CDC, phagocytosis, opsonization, etc.
  • the B7-H3-binding molecules provided herein are especially suited for the treatment of cancers.
  • the efficacy of standard monoclonal antibody therapy depends on the FcyR polymorphism of the subject (Cartron, G. et al.
  • B7-H3-binding molecules provided herein comprising a variant Fc Domain that exhibits enhanced affinity to FcyRIIIA (relative to a wild-type Fc Domain) on effector cells are better immunotherapy reagents for patients regardless of their FcyR polymorphism.
  • a combination of a B7-H3-binding molecule and a CTLA-4-binding molecule may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with cancer or other disease, or disorder by administering to a subject an effective amount of such a combination, or pharmaceutical composition(s) comprising the same.
  • the cancer is preferably a B7-H3 expressing cancer.
  • the term “combination” refers to the use of more than one therapeutic agent (e.g., a B7-H3-binding molecule and a CTLA-4-binding molecule).
  • the use of the term “combination” does not restrict the order in which therapeutic agents are administered to a subject with a disorder, nor does it mean that the agents are administered at exactly the same time, but rather it is meant that the agents are administered to a subject in a sequence and within a time interval such that the agents can act to provide an increased benefit than if they were administered otherwise.
  • each therapeutic agent e.g., (i.e., a B7-H3-binding molecule and a CTLA-4-binding molecule) may be administered at the same time or sequentially in any order and/or at different points in time so as to provide the desired therapeutic or prophylactic effect.
  • each agent need not be administered for the entire course of treatment.
  • both agents may be administered for a period of time, after which one agent is discontinued.
  • Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route, e.g., one by the oral route and one parenterally.
  • a variety of delivery systems and administration routes for providing a combination of a B7-H3-binding molecule and a CTLA-4-binding molecule are available. Delivery systems that can be used to administer a B7-H3-binding molecule and a CTLA-4-binding molecule, include, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) "Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, " J. Biol. Chem.
  • Methods of administering a B7-H3-binding molecule and a CTLA-4-binding molecule include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural e.g., epidural and mucosal
  • mucosal e.g., intranasal and oral routes.
  • molecules are administered intramuscularly, intravenously, or subcutaneously.
  • compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent (see, e.g., U.S. Patent Nos.
  • Treatment of a subject with a therapeutically or prophylactically effective amount of a B7-H3-binding molecule and/or a CTLA-4-binding molecule can include a single treatment or, preferably, can include a series of treatments.
  • a subject may be treated with a B7-H3-binding molecule and/or a CTLA-4-binding molecule once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months for between about 2 to about 52 weeks.
  • the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment.
  • the B7-H3-binding molecule and the CTLA-4-binding molecule need not be administered at the same time or at the same intervals or for the same number of treatments.
  • the B7-H3-binding molecule and the CTLA-4-binding molecule are administered using a treatment regimen comprising one or more doses, wherein the treatment regimen is administered over 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 or more than 8 weeks.
  • the treatment regimen comprises intermittently administering doses of the effective amount of such molecules (for example, administering a dose on week one and week four and not administering doses of the molecule on week two or week three).
  • the dosage of a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules, administered to a patient is typically at least about at least about 1.0 mg/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, at least about 15 mg/kg body weight, or at least about 20 mg/kg.
  • the dosage administered to a patient is typically 1.0 mg/kg to 20 mg/kg of the patient' s body weight.
  • the dosage administered to a patient is between 1.0 mg/kg and 20 mg/kg, 1.0 mg/kg and 10 mg/kg, 1.0 mg/kg and 5 mg/kg, 2.0 mg/kg and 20 mg/kg, or 5 mg/kg and 20 mg/kg of the patient' s body weight. In one embodiment, the dosage administered to a patient is between 1 mg/kg and 15 mg/kg body weight.
  • the dosage administered to a patient is 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg body weight.
  • the calculated dose will be administered based on the patient' s body weight at baseline. Significant (> 10%) change in body weight from baseline or established plateau weight should prompt recalculation of dose.
  • a fixed dosage of a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules is administered to a patient regardless of body weight.
  • the fixed dosage administered to a patient is typically between 50 mg to 500 mg.
  • the fixed dosage administered to a patient is between 50 mg and 300 mg, 100 mg and 300 mg, or 100 mg and 200 mg.
  • the fixed dosage administered to a patient is 100 mg, 200 mg or 300 mg.
  • a first therapeutic agent e.g. , anti-B7-H3 antibody or anti- CTLA-4 antibody
  • a first therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second (or subsequent) therapeutic agent (e.g., anti-B7-H3 antibody or anti-CTLA-4 antibody) to a subject with a disorder.
  • two or more agents are administered within the same patient visit
  • the B7-H3-binding molecule and the CTLA-4-binding molecule may be administered at different dosages, different concentrations, different times and/or on different schedules.
  • a dose of an anti-B7-H3 antibody is administered weekly in combination with a dose of an anti-CTLA-4 antibody administered every three or twelve weeks (wherein each administration of such a combination treatment regimen is herein referred to as a "cycle").
  • a cycle each administration of such a combination treatment regimen is herein referred to as a "cycle".
  • 1 to 15 mg (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg) of an anti-B7-H3 antibody per kg of patient body weight is administered weekly
  • 1 to 15 mg (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg) of an anti-CTLA-4 antibody per kg patient body weight is administered once every three weeks until remission of disease or unmanageable toxicity is observed.
  • treatment is divided into two periods, an "Induction Period” during which a dose of an anti-B7-H3 antibody is administered weekly in combination with a dose of an anti-CTLA-4 antibody administered every three weeks, followed by a "Maintenance Period” during which a dose of an anti-B7- H3 antibody is administered weekly alone (i.e., no further doses of an anti-CTLA-4 antibody are administered), or in combination with a dose of an anti-CTLA-4 antibody administered every twelve weeks.
  • an “Induction Period” during which a dose of an anti-B7-H3 antibody is administered weekly in combination with a dose of an anti-CTLA-4 antibody administered every three weeks
  • a "Maintenance Period” during which a dose of an anti-B7- H3 antibody is administered weekly alone (i.e., no further doses of an anti-CTLA-4 antibody are administered), or in combination with a dose of an anti-CTLA-4 antibody administered every twelve weeks.
  • an anti-B7-H3 antibody is administered to the subject by IV infusion weekly and an anti-CTLA-4 antibody is administered to the subject by IV infusion every three weeks for an Induction Period having a duration of at least 1 month or more, at least 3 months or more, or at least 6 months or more, or at least 12 months or more.
  • an anti-B7-H3 antibody is administered to the subject by IV infusion weekly alone, or in combination with an anti-CTLA-4 antibody administered to the subject by IV infusion every twelve weeks for a Maintenance Period having a duration of at least 1 month or more, at least 3 months or more, or at least 6 months or more, or at least 12 months or more.
  • IV administration once every three weeks (or twelve weeks during a Maintenance Period) an anti-B7-H3 antibody and an anti CTLA-4 antibody may be administered together or sequentially.
  • an anti-B7-H3 antibody and an anti-CTLA-4 antibody are administered to the subject sequentially by IV infusion no more than 48 hours apart.
  • an anti-B7-H3 antibody may be administered prior to, or subsequent to the administration of an anti-CTLA-4 antibody.
  • a treatment regimen may thus comprise, an Induction Period comprising 1 cycle, at least 2 cycles or more than 2 cycles, at least 3 cycles or more than 3 cycles, at least 4 cycles or more than 4 cycles, at least 5 cycles or more than 5 cycles, or at least 6 cycles or more than 6 cycles, and a an Maintenance Period comprising 1 cycle, at least 2 cycles or more than 2 cycles, at least 3 cycles or more than 3 cycles, at least 4 cycles or more than 4 cycles, at least 5 cycles or more than 5 cycles, or at least 6 cycles or more than 6 cycles .
  • the dosage of each antibody in each such cycle may be the same or may vary from the prior administered dosage, the length of each cycle may be the same or may vary from the previous cycle, and each antibody need not be administered over the entire course of treatment.
  • the antibodies not be administered as an IV push or bolus, but rather that such administration be accomplished by IV infusion.
  • the antibodies are thus preferably diluted into an infusion bag comprising a suitable diluent, e.g., 0.9% sodium chloride. Since infusion or allergic reactions may occur, premedication for the prevention of such infusion reactions is recommended and precautions for anaphylaxis should be observed during the antibody administration. It is particularly preferable for the IV infusion to be administered to the subject over a period of between 30 minutes and 24 hours.
  • the IV infusion is preferably delivered over a period of 30-180 minutes, or 30- 120 minutes, or 30-90 minutes, or over a period of 60 minutes, or over a lesser period, if the subject does not exhibit signs or symptoms of an adverse infusion reaction.
  • a preferred method of treating cancer comprising administering to a subject in need thereof a combination of an anti-B7-H3 antibody and an anti-CTLA-4 antibody in an Induction Period, followed by a Maintenance Period, wherein:
  • the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight ⁇ i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) every three weeks, such that a total of four doses of the anti-CTLA-4 antibody is administered and a total of no more than 10 doses of the anti-B7-H3 antibody is administered, and
  • the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, without any administration of the anti-CTLA-4 antibody; or
  • the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) once every twelve weeks, for the duration of such Maintenance Period.
  • the dosage of the anti-B7-H3 antibody is 1, 3, 10, or 15 mg/kg body weight. In a further embodiment, the dosage of the anti-CTLA-4 antibody is 1, 3, 6, 10, or 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight.
  • the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight.
  • the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody is 15 mg/kg body weight.
  • the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight.
  • the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight.
  • the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In any of the above embodiments, the anti-B7-H3 antibody and the anti-CTLA-4 antibody are administered by IV infusion, and when administered in the same week, both may be administered within a 48 hour, preferably 24-hour period.
  • the anti-B7-H3 antibody comprises the CDRLI CDRL2, CDRL3, CDRHI , CDRH2, and CDR H 3 Domains of hBRCA84D, hBRCA69D, PRCA157, or the CDRL I CDRL2, CDRL3, CDRHI , CDRH2, and CDR H 3 Domains of any of the anti-B7-H3 antibody provided in Table 5, and the anti-CTLA-4 antibody comprises the CDRLI CDRL2, CDRL3, CDRHI , CDRH2, and CDR H 3 Domains of CTLA-4 mAb 1 , CTLA-4 mAb 2, or the CDRLI CDRL2, CDRL3, CDRH I, CDRH2, and CDR H 3 Domains of any of the anti-CTLA-4 antibodies provided in Table 6.
  • the anti-B7-H3 antibody be hBRCA84D-2 and that the anti-CTLA-4 antibody be selected from the antibodies provided in Table 6.
  • the anti-B7-H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is ipilimumab.
  • the anti-B7-H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is tremelimumab.
  • the therapeutic agents are preferably cyclically administered to a subject. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improves the efficacy of the treatment.
  • Exemplary cycles are about once every week, about once every 10 days, about once every two weeks, and about once every three weeks.
  • Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest, etc.
  • the number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.
  • a preferred dosage regimen for the therapeutic administration as provided in any of the embodiments above comprises administering an anti-B7-H3 antibody and an anti-CTLA-4 antibody to a recipient subject in an initial cycle, and one or more subsequent cycles.
  • the anti-B7-H3 antibody and the anti-CTLA-4 antibody are administered on the same cycle schedule.
  • each cycle preferably comprises three weeks, with the anti-B7-H3 antibody being provided to the subject in a weekly administration and the anti-CTLA-4 antibody being provided to the subject on the first week of each 3 -week period.
  • each cycle for the anti-CTLA-4 antibody will preferably comprise three weeks, with the anti-CTLA-4 antibody being provided to the subject on the first week of each 3 -week period.
  • the initial cycle for the anti-B7-H3 antibody will preferably comprise 8 weeks, with the anti-B7- H3 antibody being provided to the subject in a weekly administration for the first four weeks of such 8-week initial period, followed by no administration to the subject for the period of weeks 5-8 of such 8-week initial period.
  • Each subsequent cycle will preferably comprise a 4- week period, with the anti-B7-H3 antibody being provided to the subject in a weekly administration for the first three weeks of such 4-week subsequent period, followed by no administration to the subject for the period of week 4 of such 4-week subsequent period.
  • a subject would receive the B7-H3 antibody weekly in week 1, 2, 3, 4, 9, 10, 11, 13, 14, 15, etc., wherein weeks 1-8 are the initial dosage regimen cycle, weeks 9-12 are the first subsequent cycle, weeks 13-16 are a second subsequent cycle, etc.
  • the subject would receive the CTLA-4 antibody every three weeks for a total of four doses ⁇ i.e., weeks 3, 6, 9 and 12). Accordingly: (I) during the Induction Period:
  • the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for 4 weeks (e.g., first administration cycle being weeks 1-4), followed by a 4-week suspension of such administration ⁇ e.g., in weeks 5-8), followed by a 2-week resumption of such administration ⁇ e.g., in weeks 9-10, such weeks being the first two weeks of the second administration cycle), and
  • the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight ⁇ i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) every three weeks ⁇ e.g., in weeks 1, 4, 7 and 10), such that a total of four doses of the anti-CTLA-4 antibody is administered, and
  • the anti-CTLA-4 antibody is not administered, and the anti-B7-H3 antibody is administered in one or more four week cycles at a dosage of 1 to 15 mg/kg body weight ⁇ i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for the first three weeks of a cycle, and with no administration of the anti-B7-H3 antibody in the fourth week of each cycle; or
  • the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight ⁇ i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) once every twelve weeks, for the duration of such Maintenance Period, and the anti-B7-H3 antibody is administered in three or more cycles, each cycle being of four week duration, at a dosage of 1 to 15 mg/kg body weight ⁇ i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for the first three weeks of a cycle, and with no administration of the anti-B7-H3 antibody in the fourth week of each cycle.
  • the invention further encompasses administering to a subject, a combination of a molecule that specifically binds to B7-H3 and a molecule that specifically binds to CTLA-4 in further combination with other therapies known to those skilled in the art for the treatment or prevention of cancer, autoimmune disease, inflammation, or infectious disease, including but not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery.
  • the combination of a B7-H3-binding molecule and a CTLA-4-binding molecule are administered in combination with a therapeutically or prophylactically effective amount of one or more additional therapeutic agents known to those skilled in the art for the treatment and/or prevention of cancer, in particular a B7-H3 -expressing cancer.
  • a B7-H3-binding molecule and/or a CTLA-4-binding molecule is conjugated to, or administered in further combination with, another therapeutic agent, such as, but not limited to, an alkylating agent (e.g., mechlorethamine or cisplatin), angiogenesis inhibitor, anthracycline (e.g., daunorubicin/daunomycin or doxorubicin), antibiotic (e.g., dactinomycin, bleomycin, or anthramycin), antibody (e.g., an anti-VEGF antibody such as bevacizumab (sold as AVASTIN® by Genentech, Inc.), an anti-EGFR antibody such as panitumumab (sold as VECTIBIXTM by Amgen, Inc.), or an anti-integrin antibody
  • an alkylating agent e.g., mechlorethamine or cisplatin
  • angiogenesis inhibitor e.g., anthr
  • angiogenesis inhibitors include ABT-627; angiostatin (plasminogen fragment); angiozyme; anti angiogenic antithrombin III; Bay 12- 9566; benefin; bevacizumab; BMS-275291; bisphosphonates; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagen XVIII fragment); farnesyl transferase inhibitors (FTI); fibronectin fragment; gro- beta; halofuginone; heparinases; heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); EVI-862; interferon alpha/beta/gamma; interferon inducible protein (IP- 10); interleukin-12; kringle 5 (plasminogen fragment); marimastat; metalloproteinas
  • Non-limiting examples of additional antibodies for the treatment of a cell proliferative disorder include antibodies to 17-1 A, ⁇ 3 , AFP, CD3, CD18, CD20, CD22, CD33, CD44, CD52, CEA, CTLA-4, DNA-associated proteins, EGF receptor, Ep-CAM, GD2- ganglioside, gp Illb/IIIa, gp72, HLA-DR 10 beta, HLA-DR antigen, IgE, ganglioside GD3, MUC-1, nuC242, PEM antigen, SK-1 antigen, tumor antigen CA125, tumor antigen MUCl, VEGF, and VEGF-receptor.
  • a dose escalation study is performed to determine the Maximum Tolerated Dose (MTD) or Maximum Administered Dose (MAD) (if no MTD is defined) of escalating doses of hBRCA84D-2 administered in combination with a dose of 3 mg/kg ipilimumab. This may be followed by a cohort expansion phase to further define the safety and initial efficacy of the combination with the hBRCA84D-2 dose established in the dose escalation study.
  • MTD Maximum Tolerated Dose
  • MAD Maximum Administered Dose
  • the dosing portion of the study comprises two distinct periods consisting of: 1) an Induction Period, and 2) a Maintenance Period.
  • Figure 1 provides a schematic of the schedule of administration during the Induction and Maintenance Periods.
  • the Induction Period constitutes the time when hBRCA84D-2 is administered in combination with ipilimumab. During the Induction Period hBRCA84D-2 is administered weekly and ipilimumab is administered once every 3 weeks. Once every three weeks the agents are administered on the same day with ipilimumab administered first, followed by hBRCA84D- 2. However, where the combined dose exceeds 2,500 mg the antibodies should be administered on consecutive days.
  • ipilimumab When administering the antibodies on consecutive days ipilimumab may be administered on the first day, and hBRCA84D-2 may be administered on the next calendar day. Each cycle of therapy is defined as 3 weeks, in which hBRCA84D-2 is given on Days 1, 8 and 15, and ipilimumab is given on Day 1.
  • the Induction Periods ends after a total of four doses of ipilimumab have been administered.
  • the Maintenance Period begins after the completion of the Induction period and constitutes the time when hBRCA84D-2 is administered without additional doses of ipilimumab. During the Maintenance Period hBRCA84D-2 is administered weekly.
  • hBRCA84D-2 may be evaluated in three sequential escalating doses, 3 mg/kg body weight, 10 mg/kg, and 15 mg/kg body weight, in combination with 3 mg/kg ipilimumab in cohort's patients. If it is determined that the MTD is exceeded in the first dose cohort, a dose de-escalation cohort to evaluate a lower dose of ipilimumab (1 mg/kg) in combination with 3, 10, or 15 mg/kg hBRCA84D-2 ipilimumab may be utilized.
  • Tumor assessments may be performed during the study, preferably at weeks 9, 18, 27, and every twelve weeks thereafter (i.e., weeks 39 and 51).

Abstract

The present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation. The invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers. The invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.

Description

Title of the Invention:
Combination Therapy for the Treatment of Cancer
Cross-Reference to Related Applications:
[0001] This application claims priority to U.S. Patent Appln. Serial No. 62/239,012 (filed October 8, 2015; pending), which application is herein incorporated by reference in its entirety.
Reference to Sequence Listing:
[0002] This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: 1301_128PCT_ST25.txt, created on September 26, 2016, and having a size of 67,654 bytes), which file is herein incorporated by reference in its entirety.
Field of the Invention:
[0003] The present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation. The invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers. The invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
Background of the Invention:
[0004] The growth and metastasis of tumors depends to a large extent on their capacity to evade host immune surveillance and overcome host defenses. Most tumors express antigens that can be recognized to a variable extent by the host immune system, but in many cases, an inadequate immune response is elicited because of the ineffective activation of effector T cells (Khawli, L.A. et al. (2008) "Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors," Exper. Pharmacol. 181 :291-328). A. B7 Superfamily and B7-H3
[0005] B7 family members are immunoglobulin superfamily members with an immunoglobulin- V-like and an immunoglobulin-C-like domain (e.g., IgV-IgC) (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily;' Nature Rev. Immunol. 2: 116-126). The IgV and IgC domains of B7-family members are each encoded by single exons, with additional exons encoding leader sequences, transmembrane and cytoplasmic domains. The cytoplasmic domains are short, ranging in length from 19 to 62 amino acid residues and can be encoded by multiple exons (Collins, M. et al. (2005) "The B7 Family Of Immune-Regulatory Ligands " Genome Biol. 6:223.1-223.7). Members of the B7 family are predicted to form back-to-back, non-covalent homodimers at the cell surface, and such dimers have been found with respect to B7-1 (CD80) and B7-2 (CD86). B7-1 (CD80) and B7-2 (CD86) exhibit dual specificity for the stimulatory CD28 receptor and the inhibitory CTLA-4 (CD 152) receptor (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily;' Nature Rev. Immunol. 2: 116-126).
[0006] B7-H3 is unique in that the major human form contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC) (Collins, M. et al. (2005) "The B7 Family Of Immune-Regulatory Ligands," Genome Biol. 6:223.1-223.7). Although initially thought to comprise only 2 Ig domains (IgV-IgC) (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production;' Nature Immunol. 2:269-274; Sun, M. et al. (2002) "Characterization of Mouse and Human B7-H3 Genes," J. Immunol. 168:6294- 6297), a four immunoglobulin extracellular domain variant ("4Ig-B7-H3") has been identified and found to be the more common human form of the protein (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126). However, the natural murine form (2Ig) and the human 4Ig form exhibit similar function (Hofmeyer, K. et al. (2008) "The Contrasting Role OfB7-H3," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278). The 4Ig- B7-H3 molecule inhibits the natural killer cell-mediated lysis of cancer cells (Castriconi, R. et al. "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis," Proc. Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645). The human B7-H3 (2Ig form) has been found to promote T-cell activation and IFN-γ production by binding to a putative receptor on activated T cells (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production " Nature Immunol. 2:269-274; Xu, H. et al. (2009) "MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors " Cancer Res. 69(15):5275-6281). Both B7-H4 and B7-H1 are potent inhibitors of immune function when expressed on tumor cells (Flies, D.B. et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity " J. Immunother. 30(3):251-260).
[0007] The mode of action of B7-H3 is complex, as the protein mediates both T cell co- stimulation and co-inhibition (Hofmeyer, K. et al. (2008) "The Contrasting Role OfB7-H3 " Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278; Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation And Anti-Tumor Immunity " Semin. Cancer Biol. 17(4):288-298; Subudhi, S.K. et al. (2005) "The Balance Of Immune Responses: Costimulation Verse Coinhibition " J. Mol. Med. 83 : 193-202). B7-H3 binds to (TREM)-like transcript 2 (TLT-2) and co-stimulates T cell activation, but also binds to as yet unidentified receptor(s) to mediate co-inhibition of T cells. In addition, B7-H3, through interactions with unknown receptor(s) is an inhibitor for natural killer cells and osteoblastic cells (Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278). The inhibition may operate through interactions with members of the major signaling pathways through which T cell receptor (TCR) regulates gene transcription {e.g., NFTA, NF-κΒ, or AP- 1 factors).
[0008] B7-H3 co-stimulates CD4+ and CD8+ T-cell proliferation. B7-H3 also stimulates IFN-γ production and CD8+ lytic activity (Chapoval, A. etal. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production " Nature Immunol. 2:269-274; Sharpe, A H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126). However, the protein also possibly acts through NFAT (nuclear factor for activated T cells), NF-KB (nuclear factor kappa B), and AP-1 (Activator Protein- 1) factors to inhibit T-cell activation (Yi. K.H. etal. (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4 " Immunol. Rev. 229: 145-151). B7-H3 is also believed to inhibit Thl, Th2, or Thl7 in vivo (Prasad, D.V. etal. (2004) "Murine B7-H3 Is A Negative Regulator Of T Cells " J. Immunol. 173 :2500-2506; Fukushima, A. et al. (2007) "B7-H3 Regulates The Development Of Experimental Allergic Conjunctivitis In Mice " Immunol. Lett. 113 :52-57; Yi. K.H. et al. (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4 " Immunol. Rev. 229: 145-151). Several independent studies have shown that human malignant tumor cells exhibit a marked increase in expression of B7-H3 protein and that this increased expression was associated with increased disease severity (Zang, X. et al. (2007) "The B7 Family And Cancer Therapy: Costimulation And Coinhibition " Clin. Cancer Res. 13 :5271-5279), suggesting that B7-H3 is exploited by tumors as an immune evasion pathway (Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278).
[0009] Molecules that block the ability of a B7 molecule to bind to a T-cell receptor {e.g., CD28) inhibit the immune system and have been proposed as treatments for autoimmune disease (Linsley, P S. et al. (2009) "The Clinical Utility Of Inhibiting CD28-Mediated Co- Stimulation " Immunolog. Rev. 229:307-321). Neuroblastoma cells expressing 4Ig-B7-H3 treated with anti-4Ig-B7-H3 antibodies were more susceptible to NK cells. However, it is unclear whether this activity can be attributed to only antibodies against the 4Ig-B7-H3 form because all reported antibodies raised against the 4Ig-B7-H3 also bound the two Ig-like form of B7H3 (Steinberger, P. et al. (2004) "Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains " J. Immunol. 172(4): 2352-2359 and Castriconi et al. (2004) "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis " Proc. Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645).
[0010] B7-H3 is not expressed on resting B or T cells, monocytes, or dendritic cells, but it is induced on dendritic cells by IFN-γ and on monocytes by GM-CSF (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126). The receptor(s) that bind B7-H3 have not been fully characterized. Early work suggested one such receptor would need to be rapidly and transiently up-regulated on T cells after activation (Loke, P. et al. (2004) "Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T Cells " Arthritis Res. Ther. 6:208-214). Recently, the (TREM)-like transcript 2 (TLT-2, or TREML2) receptor (King, R.G. et al. (2006) "Tr em-Like Transcript 2 Is Expressed On Cells Of The Myeloid/Granuloid And B Lymphoid Lineage And Is Up-Regulated In Response To Inflammation " J. Immunol. 176:6012-6021; Klesney-Tait, J. et al. (2006) "The TREM Receptor Family And Signal Integration " ' Nat. Immunol. 7: 1266-1273; Yi. K.H. et al. (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4," Immunol. Rev. 229: 145- 151), which is expressed on myeloid cells has been shown to be capable of binding B7-H3, and of thereby co-stimulating the activation of CD8+ T cells in particular (Zang, X. et al. (2003) "B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation " Proc. Natl. Acad. Sci. (U.S.A.) 100: 10388-10392; Hashiguchi, M. et al. (2008) "Triggering Receptor Expressed On Myeloid Cell-Like Transcript 2 (TLT-2) Is A Counter-Receptor For B7-H3 And Enhances T Cell Responses," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10495-10500; Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278).
[0011] In addition to its expression on neuroblastoma cells, human B7-H3 is over- expressed in a wide range of cancers and cultured cancer stem-like cells. In particular, B7-H3 is broadly overexpressed on many malignant neoplasms including: SCCHN, where the level is directly proportional to the development of distal metastases and decreased survival (Katayama, A., et al. (2011) "Expression of B7-H3 In Hypopharyngeal Squamous Cell Carcinoma As A Predictive Indicator For Tumor Metastasis And Prognosis," Int J Oncol 38: 1219-26); bladder cancer (Boorjian, S.A., et al. (2008) "T Cell Coregulatory Molecule Expression in Urothelial Cell Carcinoma: Clinicopathologic Correlations and Association with Survival " Clin Cancer Res 14:4800-7); prostate cancer, where expression of B7-H3 is associated with metastatic behavior and poor outcome (Chavin, G., et al. (2009) "Expression of immunosuppresive B7-H3 Ligand By Hormone-Treated Prostate Cancer Tumors And Metastases " Clin Cancer Res 15:2174-80; Zang, X., et al. (2007) "B7-H3 And B7x Are Highly Expressed In Human Prostate Cancer And Associated With Disease Spread And Poor Outcome " Proc Natl Acad Sci U S A 104: 19458-63); renal cell carcinoma, where B7-H3 is broadly expressed in tumor vasculature (Crispen, P.L., et al. (2008) "Tumor cell And Tumor Vasculature Expression OfB7-H3 Predict Survival In Clear Cell Renal Cell Carcinoma " Clin Cancer Res 14:5150-7); ovarian cancer (Zang, X., et al. (2010) "Tumor Associated Endothelial Expression OfB7-H3 Predicts Survival In Ovarian Carcinomas " Mod Pathol 2010 May 21); colorectal cancer (Sun, J., et al. (2010) "Clinical Significance And Regulation Of The Costimulatory Molecule B7-H3 In Human Colorectal Carcinoma " Cancer Immunol Immunother, Mar 24); gastric cancer (Wu, CP., et al. (2006) "Relationship Between Costimulatory Molecule B 7-H3 Expression And Gastric Carcinoma Histology And Prognosis " World J Gastroenterol 12:457-9); non-small cell lung cancer, where higher levels on the primary tumor are associated with a higher likelihood of metastatic disease (Sun, Y., et al. (2006) "B7-H3 and B7-H4 Expression In Non-Small-Cell Lung Cancer " Lung Cancer 53 : 143- 51); glioblastoma (Modak, S., et al. (2001) "Monoclonal Antibody 8H9 Targets A Novel Cell Surface Antigen Expressed By A Wide Spectrum Of Human Solid Tumors " Cancer Res 61 :4048-54); melanoma, where higher levels are associated with higher tumor stage and shorter survival (Tekle, C, etal. (2012) "B7-H3 Contributes To The Metastatic Capacity Of Melanoma Cells By Modulation Of Known Metastasis-Associated Genes " Int J Cancer 130:2282-90; Wang, L., et al. (2013) "B7-H3 Mediated Tumor Immunology: Friend Or Foe? " Int J Cancer Sep 7); and certain small round blue cell tumors of childhood including neuroblastoma and rhabdomyosarcoma (Gregorio, A., et al. (2008) "Small Round Blue Cell Tumours: Diagnostic And Prognostic Usefulness Of The Expression OfB7-H3 Surface Molecule," Histopathology 53 :73-80).
B. CTLA-4
[0012] Cytotoxic T-lymphocyte associated protein-4 (CTLA-4; CD 152) is a single pass type I membrane protein that forms a disulfide linked homo-dimer (Schwartz J.C., et al. (2001) "Structural Basis For Co-Stimulation By The Human CTLA-4/B7-2 Complex " Nature 410:604-608). Alternate splice variants, encoding different isoforms, have been characterized including a soluble isoform which functions as a monomer (Magistrelli G., et al. (1999) "A Soluble Form Of CTLA-4 Generated By Alternative Splicing Is Expressed By Nonstimulated Human T Cells " Eur J Immunol 29:3596-3602; Oaks M.K. et al. (2000) "A native soluble form of CTLA-4," Cell Immunol 2000;201 : 144-153).
[0013] CTLA-4 is primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalization (Alegre M-L, et al, (1996) "Regulation Of Surface And Intracellular Expression Of CTLA 4 On Mouse T Cells," J. Immunol. 157:4762-4770; Linsley, P.S., et al. (1996) "Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement," Immunity 4:535-543). CTLA- 4 is expressed at low levels on the surface of naive effector T cells (Alegre, M.L., et al. (1996) "Regulation Of Surface And Intracellular Expression Of CTLA4 On Mouse T Cells," J Immunol 157:4762-70), and constitutively expressed on T regulatory cells (Wang, X.B., et al.
(2002) "Expression Of CTLA-4 By Human Monocytes," Scand. J. Immunol. 55:53-60).
[0014] The extracellular region of CTLA-4 comprises a single extracellular Ig(V) domain, followed by a transmembrane (TM) region and a small intracellular cytoplasmic tail (37 amino acids). The intracellular tail contains two tyrosine-based motifs, which interact with several intracellular proteins, including the lipid kinase phosphatidylinositol 3 -kinase (PI3K), the phosphatases SHP-2 and PP2A and clathrin adaptor proteins AP-1 and AP-2 (Rudd, C.E. et al.
(2003) "Unifying Concepts In CD28, ICO S And CTLA4 Co-Receptor Signalling," Nat Rev Immunol. 3 : 544-56). CTLA-4 is related to CD28, with the two proteins having approximately 29% identity at the amino acid level (Harper, K. (1991) "CTLA-4 And CD28 Activated Lymphocyte Molecules Are Closely Related In Mouse And Human As To Sequence, Message Expression, Gene Structure, And Chromosomal Location," J. Immunol. 147: 1037-1044). [0015] When a naive T effector cell is activated through its T cell receptors (TCRs), CTLA- 4 is recruited to the cell surface (Linsley, P.S., etal. (1996) "Intracellular Trafficking Of CTLA- 4 And Focal Localization Towards Sites Of TCR Engagement," Immunity 4:535-43). Once CTLA-4 is expressed on the T cell surface, it competes with CD28 (constitutively expressed on T cells) for CD80/CD86, thereby shutting off further signaling through the TCR and thus down-regulating any further T cell response by TCR signaling (Carreno, B.M., et al. (2000) "CTLA-4 (CD 152) Can Inhibit T Cell Activation By Two Different Mechanisms Depending On Its Level Of Cell Surface Expression " J Immunol 165: 1352-6;Chuang, E., et al. (1999) "Regulation Of Cytotoxic T Lymphocyte-Associated Molecule-4 By Src Kinases " J Immunol 162: 1270-7). Thus, CTLA-4 acts as a negative regulator of T effector cell activation that diminishes effector function and dictates the efficacy and duration of a T cell response (Linsley, P.S., et al. (1996) "Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement ," Immunity 4:535-43).
[0016] In addition, CTLA-4 may play a role in enhancing the negative effect of regulatory T cells on the immune response to cancer (Tai, Y.T., et al., (2012) "Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HM1.24 Antibody Against Multiple Myeloma via Augmented Effector Function," Blood 119:2074-82). CTLA-4 has a much higher affinity for members of the B7 family than for CD28, and therefore its expression on a T effector cell dictates a dominant negative regulation of the T cell (Allison, J.P., et al. (1995) "Manipulation Of Costimulatory Signals To Enhance Antitumor T-Cell Responses," Curr Opin Immunol 7:682-6). The mechanism by which CTLA-4 contributes to the suppressor function of T regulatory cells is incompletely understood, but the expression of CTLA-4 on T regulatory cells enhances the suppressive function of these cells (Tai, Y.T., et al, (2012) "Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HMl .24 Antibody Against Multiple Myeloma via Augmented Effector Function," Blood 119:2074-82).
C. B7-H3-Expressing Cancers
[0017] Overexpression of B7-H3 occurs in a wide range of cancers and cultured cancer stem-like cells. As stated above, the overexpression of B7-H3 is strongly associated with increased disease recurrence and a poor prognosis. However, B7-H3 is a potential target for anti-B7-H3 drugs, including monoclonal antibodies that target the extracellular domain of the receptor. Antibodies and other molecules that specifically bind to B7-H3 have been described (see, United States Patent No. 8,802,091, 7,527,969; 7,368,554; 7,358,354; and 7,279,567; United States Patent Application Publications Nos. US 20090087416; US 20090022747; US 20090018315; US2008116219; US20080081346; US 20050202536; US20030103963; US20020168762; PCT Publications Nos. WO 2011/109400; WO 2008/116219; WO 2006/016276; WO 2004/093894; WO 04/001381; WO 2002/32375; WO 2002/10187 and WO 2001/094413; EP 1292619B; Modak, S. et al. (March 1999) "Disialoganglioside GD2 And Antigen 8H9: Potential Targets For Antibody-Based Immunotherapy Against Desmoplastic Small Round Cell Tumor (DSRCT) And Rhabdomyosarcoma (RMS)," Proceedings Of The American Association For Cancer Research Annual Meeting, Vol. 40:474 (90th Annual Meeting Of The American Association For Cancer Research; Philadelphia, Pennsylvania, US; April 10-14, 1999; Modak, S. et al. (March 2000) "Radioimmunotargeting To Human Rhabdomyosarcoma Using Monoclonal Antibody 8H9 " Proc. Am. Assoc. Cancer Res.41 :724; Modak, S. et al. (2001) "Monoclonal Antibody 8H9 Targets A Novel Cell Surface Antigen Expressed By A Wide Spectrum Of Human Solid Tumors " Cancer Res. 61(10):4048-4054; Steinberger, P. et al. (2004) "Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains " J. Immunol. 172(4):2352-2359; Xu, H. et al. (2009) "MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors " Cancer Res. 69(15):5275-6281).
[0018] Despite these advances, a need remains for improved therapies for treating cancers expressing B7-H3 and of facilitating or mediating an immune response against such cancers. Although the adaptive immune system can be a potent defense mechanism against cancer and disease, it is often hampered by immune suppressive mechanisms in the tumor microenvironment, such as the expression of co-inhibitory molecules. Furthermore, co- inhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor milieu can dominantly attenuate T-cell responses against cancer cells. Thus, there exists a further need for novel combinations and treatment regimens that specifically recognize targets on the surface of cancer cells, and which can thereby mediate T-cell activation, simulation of an immune response and killing of cancer cells that express B7-H3. It is an object of this invention to identify such compositions and treatment regimens.
Summary of the Invention:
[0019] The present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation. The invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers. The invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
[0020] In detail, the invention provides a method of treating a cancer comprising administering to a subject in need thereof, a molecule that specifically binds to B7-H3, and a molecule that specifically binds to CTLA-4.
[0021] The invention particularly concerns embodiments of such methods, wherein the molecule that specifically binds to B7-H3 is an anti-B7-H3 antibody or antigen-binding fragment thereof, and the molecule that specifically binds to CTLA-4 is an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.
[0022] The invention particularly concerns the embodiment of such methods wherein the anti-B7-H3 antibody, or antigen-binding fragment thereof:
(a) competes for B7-H3 binding with BRCA84D, BRCA69D, PRCA157, or an anti-B7-H3 antibody selected from Table 5; or
(b) has the three heavy chain CDRs and the three light chain CDRs of BRCA84D, hBRCA84D (1.1), hBRCA84D (2.2), hBRCA84D-2, hBRCA69D (1.1), hBRCA (2.2), or the three heavy chain CDRs and the three light chain CDRs of an anti-B7-H3 selected from Table 5; or
(c) has the heavy chain variable domain and the light chain variable
domain of BRCA84D, hBRCA84D (1.1), hBRCA84D (2.2), hBRCA84D-2, hBRCA69D (1.1), hBRCA (2.2), or the heavy chain variable domain and the light chain variable domain of an anti-B7-H3 selected from Table 5.
[0023] The invention particularly concerns the embodiment of such methods wherein the anti-CTLA-4 antibody, or antigen-binding fragment thereof:
(a) competes for CTLA-4 binding with ipilimumab, tremelimumab, or with an anti-CTLA-4 antibody selected from Table 6; or (b) has the three heavy chain CDRs and the three light chain CDRs of ipilimumab, tremelimumab, or the three heavy chain CDRs and the three light chain CDRs of an anti-CTLA-4 antibody selected from
Table 6; or
(c) has the heavy chain variable domain and the light chain variable
domain of ipilimumab, tremelimumab, or the heavy chain variable domain and the light chain variable domain of an anti-CTLA-4 antibody selected from Table 6.
[0024] The invention further concerns embodiments of such methods wherein the anti-B7- H3 antibody or antigen-binding fragment thereof comprises an Fc Domain, and/or the anti- CTLA-4 antibody, or antigen-binding fragment thereof comprises an Fc Domain. The invention further concerns the embodiments of such methods wherein the anti-B7-H3 antibody or antigen-binding fragment thereof, comprises a variant Fc Domain having one, two, three, four, five or more substitutions in the Fc Domain that enhance(s) ADCC. The invention further concerns the embodiments of such methods wherein the Fc Domain modifications that enhance ADCC comprise any one, any two, any three, any four, or all five of the substitutions L235V, F243L, R292P, Y300L and P396L, preferably of an IgGl Fc Domain.
[0025] The invention further concerns embodiments of such methods, wherein the anti-B7- H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is ipilimumab, or tremelimumab. The invention further concerns embodiments of such methods, wherein the anti-B7-H3 antibody is hBRCA84D-2, and the anti-CTLA-4 antibody, or the antigen-binding fragment thereof, comprises the VH and VL Domains of ipilimumab, tremelimumab, or the VH and VL Domains of an anti-CTLA-4 antibody selected from Table 6.
[0026] The invention additionally concerns embodiments of such methods wherein the method comprises an Induction Period followed by a Maintenance Period, wherein:
(A) the Induction Period comprises administering to a subject in need thereof a combination of an anti-B7-H3 antibody and an anti-CTLA-4 antibody, wherein
(i) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg recipient body weight weekly; and (ii) the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg recipient body weight every three weeks for a total of four doses of the anti-CTLA-4 antibody, and
(B) the Maintenance Period comprises:
(i) administering to the subject the anti-B7-H3 antibody at a dosage of 1 to 15 mg/kg recipient body weight weekly; or
(ii) administering to the subject the anti-B7-H3 antibody at a dosage of 1 to 15 mg/kg recipient body weight weekly and administering the anti- CTLA-4 antibody at a dosage of 1 to 15 mg/kg recipient body weight every twelve weeks.
[0027] The invention also concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage of 1-15 mg/kg recipient body weight and the molecule that specifically binds to CTLA- 4 (particularly an anti-CTLA-4 antibody) is administered at a dosage of 1-15 mg/kg recipient body weight. The invention further concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage selected from 1 mg/kg, 3 mg/kg, 10 mg/kg and 15 mg/kg recipient body weight and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) is administered at a dosage selected from 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, and 15 mg/kg recipient body weight. The invention particularly concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) is administered at a dosage selected from 3 mg/kg, 10 mg/kg and 15 mg/kg recipient body weight, and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) is administered at a dosage of 3 mg/kg recipient body weight. The invention further concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) are administered by IV infusion. The invention also concerns embodiments of such methods wherein the molecule that specifically binds to B7-H3 (particularly an anti-B7-H3 antibody) and the molecule that specifically binds to CTLA-4 (particularly an anti-CTLA-4 antibody) are administered every three or twelve weeks within a 48 hour period. [0028] The invention particularly concerns embodiments of such methods wherein the cancer is a cancer in which B7-H3 is expressed. Invention further concerns embodiments of such methods wherein the cancer is a squamous cell cancer of the head and neck (SCCHN), a bladder cancer, a breast cancer, a colorectal cancer, a gastric cancer, a glioblastoma, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, a pharyngeal cancer, a prostate cancer, a renal cell carcinoma, a small round blue cell tumor, a neuroblastoma, or a rhabdomyosarcoma, in which B7-H3 is expressed.
[0029] The invention additionally concerns embodiments of such methods further comprising the step of administering a third therapeutic agent, particularly wherein the third therapeutic agent is an anti-angiogenic agent, an anti -neoplastic agent, a chemotherapeutic agent, or a cytotoxic agent
Brief Description of the Drawings:
[0030] Figure 1 provides a schematic of a combination dosing schedule having an Induction Period followed by a Maintenance Period.
Detailed Description of the Invention:
[0031] The present invention is directed to a combination therapy involving the administration of a first molecule that specifically binds to human B7-H3 and a second molecule that that specifically binds to human CTLA-4 to a subject for the treatment of cancer and/or inflammation. The invention also concerns pharmaceutical compositions that comprise a first molecule that specifically binds to human B7-H3 and a second molecule that specifically binds to human CTLA-4 that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with any of a variety of human cancers. The invention also relates to the use of such pharmaceutical compositions to treat cancer and other diseases in recipient subjects.
A. Antibodies
1. Antibodies
[0032] "Antibodies" are immunoglobulin molecules capable of immunospecific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., (an "antigen") through at least one epitope recognition site, located in the Variable Domain of the immunoglobulin molecule. Thus, whereas the target molecule is an "antigen", the portion of the antigen that is recognized by an antibody and to which the antibody binds is an "epitope." As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, camelized antibodies, single-chain antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention), but also mutants thereof, naturally occurring variants, fusion proteins comprising an epitope binding site of the required immunospecificity, humanized antibodies, and chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required immunospecificity. As used herein, an "antigen-binding fragment" of an antibody is an immunoglobulin whose amino acid sequence comprises at least one epitope-binding site of an antibody specific for such antigen. As used herein, the term encompasses fragments (e.g., Fab, Fab', F(ab')2 Fv), disulfide-linked bispecific Fvs (sdFv), intrabodies, and single-chain molecules (e.g., scFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an epitope-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
[0033] Natural antibodies (such as IgG antibodies) are composed of two Light Chains complexed with two Heavy Chains. Each Light Chain contains a Variable Light Chain (VL) Domain and a Constant Light Chain (CL) Domain. Each heavy chain contains a Variable Heavy Chain (VH) Domain, three Heavy Chain Constant Domains (CHI, CH2 and CH3), and a Hinge Domain, which is located between the CHI and CH2 Domains. The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is thus a tetramer having two light chains and two heavy chains, usually expressed as a glycoprotein of about 150,000 Da. The amino-terminal portion of each polypeptide chain includes its Variable ("V") Domain, which is of about 100 to 110 or more amino acids, and is primarily responsible for antigen recognition. The carboxy -terminal portion of each polypeptide chain defines the chain's Constant ("C") Domain. Thus, the structure of the light chains of an IgG molecule (in the N-terminal to C- terminal direction) is: VL-CL, and the structure of the IgG heavy chains (in the N-terminals to C-terminal direction) is: VH-CH1-Hinge-CH2-CH3.
[0034] The ability of an intact, unmodified antibody (e.g. , an IgG antibody) to bind an epitope of an antigen depends upon the presence of Variable Domains on the immunoglobulin light and heavy chains (i.e., the VL Domain and VH Domain, respectively). Interaction of an antibody Light Chain and an antibody heavy chain and, in particular, interaction of its VL and VH Domains forms one of the epitope-binding sites of the antibody. The Variable Domains of an IgG molecule consist of the complementarity determining regions (CDR), which contain the residues in contact with epitope, and non-CDR segments, referred to as framework segments (FR), which in general maintain the structure and determine the positioning of the CDR loops so as to permit such contacting (although certain framework residues may also contact antigen). Thus, the VL Domains have the structure (in the N-terminal to C-terminal direction): FRl1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRl4, and the VH Domains have the structure (in the N-terminal to C-terminal direction): FRh1-CDRH1-FRH2-CDRH2-FRh3- CDRH3-FRH4. Polypeptides that are (or may serve as) the first, second and third CDR of an antibody Light Chain are herein respectively designated CDRLI Domain, CDRL2 Domain, and CDRL3 Domain. Similarly, polypeptides that are (or may serve as) the first, second and third CDR of an antibody heavy chain are herein respectively designated CDRHI Domain, CDRH2 Domain, and CDRH3 Domain. Thus, the terms CDRLI Domain, CDRL2 Domain, CDRL3 Domain, CDRHI Domain, CDRH2 Domain, and CDRH3 Domain are directed to polypeptides that when incorporated into a protein cause that protein to be able to bind to a specific epitope regardless of whether such protein is an antibody having light and heavy chains or a single-chain binding molecule (e.g., an scFv, a BiTe, etc.), or is another type of protein. In contrast to such antibodies, the scFv construct comprises a VL and VH Domain of an antibody contained in a single polypeptide chain wherein the Domains are separated by a flexible linker of sufficient length to allow self-assembly of the two Domains into a functional epitope-binding site. Where self-assembly of the VL and VH Domains is rendered impossible due to a linker of insufficient length (less than about 12 amino acid residues), two of the scFv constructs may interact with one another other to form a bivalent molecule in which the VL of one chain associates with the VH of the other (reviewed in Marvin et al. (2005) "Recombinant Approaches To IgG-Like Bispecifw Antibodies, " Acta Pharmacol. Sin. 26:649-658).
[0035] In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. The last few decades have seen a revival of interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnology-derived drugs (Chan, C.E. et al. (2009) "The Use Of Antibodies In The Treatment Of Infectious Diseases," Singapore Med. J. 50(7):663-666). Nearly 200 antibody- based drugs have been approved for use or are under development. [0036] The term "monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non- naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term "monoclonal antibody" encompasses not only an intact monoclonal antibody and a full- length monoclonal antibody, but also a fragment thereof (such as an Fab, Fab', F(ab')2 Fv), single-chain (scFv), a mutant thereof, a fusion protein comprising an antibody portion, a humanized monoclonal antibody, a chimeric monoclonal antibody, and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site of the required immunospecificity and the ability to bind to an epitope. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody." Methods of making monoclonal antibodies are known in the art. One method which may be employed is the method of Kohl er, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity,'" Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats or rabbits. The antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that contain the desired epitope. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, proteins, peptides, nucleic acids, or tissue. Cells used for immunization may be cultured for a period of time (e.g. , at least 24 hours) prior to their use as an immunogen. Cells may be used as immunogens by themselves or in combination with a non-denaturing adjuvant, such as Ribi (see, e.g., Jennings, V.M. (1995) "Review of Selected Adjuvants Used in Antibody Production," ILAR J. 37(3): 119- 125).
[0037] In general, cells should be kept intact and preferably viable when used as immunogens. Intact cells may allow antigens to be better detected than ruptured cells by the immunized animal. Use of denaturing or harsh adjuvants, e.g., Freund's adjuvant, may rupture cells and therefore is discouraged. The immunogen may be administered multiple times at periodic intervals such as, bi weekly, or weekly, or may be administered in such a way as to maintain viability in the animal (e.g., in a tissue recombinant). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for a desired pathogenic epitope can be sequenced and produced recombinantly by any means known in the art. In one embodiment, such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such antibodies may be used for genetic manipulation to generate a chimeric antibody, a humanized antibody, or a caninized antibody, or to improve the affinity, or other characteristics of the antibody. The term "humanized" antibody refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and /or sequence of a human immunoglobulin. The polynucleotide sequence of the variable domains of such antibodies may be used for genetic manipulation to generate such derivatives and to improve the affinity, or other characteristics of such antibodies. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen- binding portion of the antibody, while swapping the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable Domains (2) designing the humanized antibody or caninized antibody, i.e., deciding which antibody framework region to use during the humanizing or canonizing process (3) the actual humanizing or caninizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Patents Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.
[0038] The epitope-binding site of such antibodies may comprise either complete Variable Domains fused onto Constant Domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the Variable Domains. Antigen-binding sites may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When non-human antibodies are prepared with respect to a particular antigen, the variable regions can be "reshaped" or "humanized" by grafting CDRs derived from non-human antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53 :851-856. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity■" Science 239: 1534-1536; Kettleborough, C. A. etal. (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation " Protein Engineering 4:773-3783; Maeda, H. et al. (1991) "Construction Of Reshaped Human Antibodies With HIV -Neutralizing Activity " Human Antibodies Hybridoma 2: 124-134; Gorman, S. D. et al. (1991) "Reshaping A Therapeutic CD4 Antibody " Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo " Bio/Technology 9:266-271; Co, M. S. et al. (1991) "Humanized Antibodies For Antiviral Therapy " Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) "Humanization Of An Anti-pl85her2 Antibody For Human Cancer Therapy " Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD 33 Antigen " J. Immunol. 148: 1149-1154. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which differ in sequence relative to the original antibody.
[0039] A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains (see, for example, Winter et al. (1991) "Man-made Antibodies " Nature 349:293-299; Lobuglio et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response " Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw et al. (1987) "Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1 A) To A Colon Cancer Tumor-Associated Antigen " J. Immunol. 138:4534-4538, and Brown et al. (1987) "Tumor -Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody " Cancer Res. 47:3577-3583). Other references describe rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain (see, for example, Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity " Science 239: 1534-1536; and Jones et al. (1986) "Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse " Nature 321 :522-525). Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, for example, European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize unwanted immunological response toward rodent anti- human antibody molecules, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. Other methods of humanizing antibodies that may also be utilized are disclosed by Daugherty et al. (1991) "Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD 18 Component Of Leukocyte Integrins," Nucl. Acids Res. 19:2471-2476 and in U.S. Patents Nos. 6, 180,377; 6,054,297; 5,997,867; and 5,866,692.
2. Bi-Specific Antibodies
[0040] Natural antibodies are capable of binding to only one epitope species {i.e., they are "mono-specific"), although they may be able to bind multiple copies of that species {i.e., they may exhibit bi-valency or multi-valency). The functionality of antibodies can be enhanced by generating multispecific antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating antibody- based molecule having higher valency {i.e., more than two binding sites) for the same epitope and/or antigen.
[0041] A wide variety of recombinant bi-specific and tri-specific antibody formats have been developed (see, e.g., United States Patent No. 8,277,806; 6,994,853; 6,551,592 and 6, 171,586; United States Patent Publication No. 2010-0291112 and 2008-0057054; and PCT Publication Nos. WO 2013/070565, WO 2012/156430, WO 2012/009544, WO 2009/132876, WO 2009/018386, WO 2008/003116, WO 2008/003103, WO 2007/146968, WO 2006/072152, WO 2002/020039, WO 2000/018806; WO 1999/042597, WO 1998/006749 and WO 1998/003670), most of which use linker peptides either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further epitope-binding site {e.g., scFv, VL VH, etc.) to, or within, the antibody core, or to fuse multiple epitope-binding site {e.g. two Fab fragments or scFv) to each other. Alternative formats use linker peptides to fuse an epitope-binding site (e.g., an scFv, VL, VH, etc.) to a dimerization domain such as the CH2-CH3 Domain or alternative polypeptides (WO 2005/070966, WO 2006/107786A WO 2006/107617A, WO 2007/046893). PCT Publications Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 teach tri- specific antibodies in which the CL and CHI Domains are switched from their respective natural positions and the VL and VH Domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind to more than one antigen. PCT Publications Nos. WO 2013/163427 and WO 2013/119903 disclose modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain. PCT Publications Nos. WO 2010/028797, WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc Domains have been replaced with additional VL and VH Domains, so as to form tri-valent binding molecules. PCT Publications No. WO 2013/006544 discloses multi-valent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to yield heterodimeric structures. PCT Publications Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose adding additional Binding Domains or functional groups to an antibody or an antibody portion (e.g., adding additional VL and VH Domains to the antibody's light and heavy chains, or adding a heterologous fusion protein or chaining multiple Fab Domains to one another). United States Patent No. 7,695,936 and Patent Publication 2007/ 0196363 concern bi-specific antibodies that are formed from the heavy chains of two antibodies, one of which possess a protuberance engineered into its heavy chain and the second of which possess a complementary cavity engineered into its heavy chain. The presence of such complementary "knobs" and "holes" is taught to preferentially form bi- specific hetero-antibodies (having one heavy chain of each such antibody) relative to monospecific homo-antibodies that contain two heavy chains of the same antibody.
3. Preferred Fc Domains
[0042] The CH2 and CH3 Domains of the two heavy chains interact to form the Fc Domain, which is a domain that is recognized by cellular Fc Receptors (FcyRs). As used herein, the term "Fc Domain" is used to define a C-terminal region of an IgG heavy chain. The amino acid sequence of the CH2-CH3 domain of an exemplary human IgGl is (SEQ ID NO:l):
231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVKFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPGX
as numbered by the EU index as set forth in Kabat, wherein X is a lysine (K) or is absent.
[0043] The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG2 is (SEQ ID NO:2):
231 240 250 260 270 280
APPVA-GPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVQFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQFNSTF RWSVLTWH QDWLNGKEYK CKVSNKGLPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE
390 400 410 420 430
WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPGX
as numbered by the EU index as set forth in Kabat, wherein, X is a lysine (K) or is absent.
[0044] The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG3 is (SEQ ID NO:3):
231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVQFKWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTF RWSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE
440 447
ALHNRFTQKS LSLSPGX
as numbered by the EU index as set forth in Kabat, wherein, X is a lysine (K) or is absent. [0045] The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG4 is (SEQ ID NO:4):
231 24 0 250 2 60 27 0 2 8 0
APE FLGGPSV FLFPPKPKDT LMI SRTPEVT CWVDVSQED PEVQFNWYVD
2 90 300 31 0 32 0 330
GVEVHNAKTK PREEQFNS TY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS
34 0 350 3 60 37 0 38 0
S IEKT I SKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
390 4 00 4 1 0 42 0 430
WESNGQPENN YKTTPPVLDS DGS FFLYSRL TVDKSRWQEG NVFSCSVMHE
44 0 447
ALHNHYTQKS LSLSLGX as numbered by the EU index as set forth in Kabat, wherein, X is a lysine (K) or is absent.
[0046] Throughout the present specification, the numbering of the residues in the constant region of an IgG heavy chain is that of the EU index as in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, H1, MD (1991), expressly incorporated herein by references. The "EU index as in Kabat" refers to the numbering of the human IgGl EU antibody. Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid. Rabat's numbering scheme is extendible to antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids. This method for assigning residue numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position to an amino acid at position 50 of a mouse antibody light chain.
[0047] Although boundaries may vary slightly, the CH2 domain of a human IgG Fc Domain usually extends from amino acids 231 to amino acid 341 of a human IgG according to the EU numbering system of Kabat. The CH3 domain of a human IgG usually extends from amino acids 342 to 447 according to the EU numbering system of Kabat. The "hinge region" or "hinge domain" is generally defined as stretching from Glu216 to Pro230 of human IgGl .
[0048] Polymorphisms have been observed at a number of different positions within antibody constant regions {e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index as set forth in Kabat), and thus slight differences between the presented sequence and sequences in the prior art can exist. Polymorphic forms of human immunoglobulins have been well-characterized. At present, 18 Gm allotypes are known: Glm (1, 2, 3, 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5) (Lefranc, et al, The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet. : 50, 199-211). It is specifically contemplated that the antibodies useful in the methods of the present invention may incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype or haplotype of the sequences provided herein. Furthermore, in some expression systems the C-terminal amino acid residue (bolded above) of the CH3 Domain may be post-translationally removed. Accordingly, the C-terminal residue of the CH3 Domain is an optional amino acid residue in the B7-H3-binding molecules and CTLA- 4-binding molecules provided herein. Specifically encompassed by the instant invention are such binding molecules lacking the C-terminal residue of the CH3 Domain. Also specifically encompassed by the instant invention are such binding molecules comprising the C-terminal lysine residue of the CH3 Domain.
[0049] When present, the CHI Domain and/or hinge region may be of any isotype (e.g., IgGl, IgG2, IgG3, or IgG4), but is preferably of the same isotype as the desired Fc Domain.
[0050] The amino acid sequence of an exemplary human IgGl CHI Domain is (SEQ ID NO:8):
AS TKGPSVFP LAPS SKS TSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HT FPAVLQS S GLYSLS SWT VPS S SLGTQT YI CNVNHKPS NTKVDKRV
[0051] The amino acid sequence of an exemplary human IgG2 CHI Domain is (SEQ ID NO:9):
AS TKGPSVFP LAPCSRS TSE S TAALGCLVK DYFPEPVTVS WNSGALTSGV HT FPAVLQS S GLYSLS SWT VPS SNFGTQT YTCNVDHKPS NTKVDKTV
[0052] The amino acid sequence of an exemplary human IgG4 CHI Domain is (SEQ ID NO:10):
AS TKGPSVFP LAPCSRS TSE S TAALGCLVK DYFPEPVTVS WNSGALTSGV HT FPAVLQS S GLYSLS SWT VPS S SLGTKT YTCNVDHKPS NTKVDKRV [0053] The amino acid sequence of an IgG4 CHI Domain and Stabilized Hinge (SEQ ID
NO:47) is shown below.
AS TKGPSVFP LAPCSRS TSE S TAALGCLVK DYFPEPVTVS WNSGALTSGV HT FPAVLQS S GLYSLS SWT VPS S SLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP
[0054] The amino acid sequence of an exemplary human IgGl hinge region is (SEQ ID NO: ll): EPKSCDKTHTCPPCP
[0055] The amino acid sequence of an exemplary human IgG2 hinge region is (SEQ ID NO: 12):
ERKCCVECPPERKCCVECPPCP
[0056] The amino acid sequence of an exemplary human IgG4 hinge region is (SEQ ID NO: 13): ESKYGPPCPSCP
[0057] Activating and inhibitory signals are transduced through the Fc gamma Receptors (FcyRs) following their ligation to an Fc Domain. These diametrically opposing functions result from structural differences among the different receptor isoforms. Two distinct domains within the cytoplasmic signaling domains of the receptor called immunoreceptor tyrosine- based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITFMS) account for the different responses. The recruitment of different cytoplasmic enzymes to these structures dictates the outcome of the FcyR-mediated cellular responses. ITAM-containing FcyR complexes include FcyRI, FcyRIIA, FcyRIIIA, whereas ITIM-containing complexes only include FcyRIIB. Human neutrophils express the FcyRIIA gene. FcyRIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation. ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates (e.g., PbK). Cellular activation leads to release of proinflammatory mediators. The FcyRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds IgG complexes in an indistinguishable manner. The presence of an ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently the molecular basis of this inhibition was established. When co-ligated along with an activating FcyR, the ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of the inositol polyphosphate 5 '-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of ITAM-containing FcyR-mediated tyrosine kinase activation, consequently preventing the influx of intracellular Ca++. Thus cross-linking of FcyRIIB dampens the activating response to FcyR ligation and inhibits cellular responsiveness. B cell activation, B cell proliferation and antibody secretion is thus aborted. In addition, interaction with the neonatal Fc Receptor (FcRn) mediates the recycling of IgG molecules from the endosome to the cell surface and release into the blood.
[0058] Modification of the Fc Domain normally leads to an altered phenotype, for example altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function. It may be desirable to modify the Fc Domain of the B7-H3 -binding molecules and/or CTLA-4-binding molecules (e.g., anti-B7-H3 and anti-CTLA-4 antibodies) for use in the methods of the present invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies whose mechanism of action involves blocking or antagonism, but not killing of the cells bearing a target antigen. Increased effector function is generally desirable when directed to undesirable cells, such as tumor and foreign cells, where the FcyRs are expressed at low levels, for example, tumor-specific B cells with low levels of FcyRIIB (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma).
[0059] The Fc Domain of the B7-H3-binding molecules and/or CTLA-4-binding molecules (e.g., anti-B7-H3 and anti-CTLA-4 antibodies) for use in the methods of the present invention may be either a complete Fc Domain (e.g., a complete IgG Fc Domain) or only a fragment of a complete Fc Domain. Thus, the Fc Domain of the molecules useful in the methods of the present invention that contain such a domain may include some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete Fc Domain, or may comprise a variant CH2 and/or a variant CH3 sequence (that may include, for example, one or more insertions and/or one or more deletions with respect to the CH2 or CH3 domains of a complete Fc Domain). Such Fc Domains may comprise non-Fc polypeptide portions, or may comprise portions of non-naturally complete Fc Domains, or may comprise non-naturally occurring orientations of CH2 and/or CH3 domains (such as, for example, two CH2 domains or two CH3 Domains, or in the N-terminal to C-terminal direction, a CH3 Domain linked to a CH2 Domain, etc.). Although the Fc Domain of the B7-H3-binding molecules and/or CTLA-4-binding molecules may possess the ability to bind to one or more Fc receptors (e.g., FcyR(s)), more preferably such Fc Domain will cause altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by a wild-type Fc Domain) or will substantially eliminate the ability of such Fc Domain to bind to one or more FcyR (e.g., inhibitory receptor(s)).
[0060] In certain embodiments, the molecules for use in the methods of the present invention may comprise a variant Fc Domain having altered affinities for an activating and/or inhibitory Fey receptor. In one embodiment, the molecules comprise a variant Fc Domain that has increased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain. In another embodiment, the molecules for use in the methods of the present invention comprise a variant Fc Domain that has decreased affinity for FcyRIIB and increased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain. In yet another embodiment, molecules for use in the methods of the present invention comprise a variant Fc Domain that has decreased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain. In still another embodiment, the molecules for use in the methods of the present invention comprise a variant Fc Domain that has unchanged affinity for FcyRIIB and decreased (or increased) affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with a wild-type Fc Domain.
[0061] In certain embodiments, the molecules for use in the methods of the present invention comprise a variant Fc Domain having an altered affinity for FcyRIIIA and/or FcyRIIA such that the immunoglobulin has an enhanced effector function. Non-limiting examples of effector cell functions include antibody dependent cell mediated cytotoxicity, antibody dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, Clq binding, and complement dependent cell mediated cytotoxicity.
[0062] Variant Fc Domains are well known in the art, and any known variant Fc Domain may be used in the present invention to confer or modify the effector function exhibited by a molecule comprising an Fc Domain (or portion thereof) as functionally assayed, e.g., in an NK dependent or macrophage dependent assay. For example, Fc Domain variants identified as altering effector function are disclosed in PCT Publications No. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; and WO 2008/140603, and any suitable variant disclosed therein may be used in the present molecules. Table 4 lists exemplary single, double, triple, quadruple and quintuple Fc Domain
Table 4
Variations of Preferred Activating Fc Domains
Single-Site Va riat ions
F243L R292G D270E R292P
Y300L P396L
Double-Site Variat ions
F243L and R292P F243L and Y300L F243L and P396L R292P and Y300L
D270E and P396L R292P and V305I P396L and Q419H P247L and N421K
R292P and P396L Y300L and P396L R255L and P396L R292P and P305I
K392T and P396L
Triple-Site Va riations
F243L, P247L and N421K P247L, D270E and N421K
F243L, R292P and Y300L R255L, D270E and P396L
F243L, R292P and V305I D270E, G316D and R416G
F243L, R292P and P396L D270E, K392T and P396L
F243L, Y300L and P396L D270E, P396L and Q419H
V284M, R292L and K370N R292P, Y300L and P396L
Quad ru ple-Site Va riat ions
L234F, F243L, R292P and Y300L F243L, P247L, D270E and N421K
L234F, F243L, R292P and Y300L F243L, R255L, D270E and P396L
L235I, F243L, R292P and Y300L F243L, D270E, G316D and R416G
L235Q, F243L, R292P and Y300L F243L, D270E, K392T and P396L
P247L, D270E, Y300L and N421K F243L, R292P, Y300L, and P396L
R255L, D270E, R292G and P396L F243L, R292P, V305I and P396L
R255L, D270E, Y300L and P396L F243L, D270E, P396L and Q419H
D270E, G316D, P396L and R416G
Qu int uple-Site Va riat ions
L235V, F243L, R292P, Y300L and P396L F243L, R292P, V305L Y300L and P396L
L235P, F243L, R292P, Y300L and P396L
Particularly preferred variants include one or more modifications selected from
Figure imgf000027_0001
Figure imgf000028_0001
[0065] Still more particularly preferred variants include one or more modifications selected from Groups 1-105:
Figure imgf000028_0002
Group Variant Group Variant
13 F241R / F243Q / V262T / V264R 66 S239D / N297D / I332E / K326E
14 F241R / F243Q / V262T / V264R / 67 S239D / N297D / I332E / L235D I332E
15 F241W / F243W / V262A / 68 S239D/ S298A/I332E
V264A
16 F241Y / F243Y / V262T / V264T 69 S239D / V264I / A330L / I332E
17 F241Y / F243Y / V262T / V264T / 70 S239D/ V264I/I332E
N297D/I332E
18 F243L / V262I / V264W 71 S239D / V264I / S298A / I332E
19 P243L / V264I 72 S239E/D265N
20 L328D / I332E 73 S239E/D265Q
21 L328E/I332E 74 S239E/I332D
22 L328H/I332E 75 S239E/I332E
23 L328I/I332E 76 S239E/I332N
24 L328M/I332E 77 S239E/I332Q
25 L328N/I332E 78 S239E/N297D/I332E
26 L328Q / I332E 79 S239E / V264I / A330Y / 1332 E
27 L328T/I332E 80 S239E/ V264I/I332E
28 L328V/I332E 81 S239E / V264I / S298A / A330Y
/I332E
29 N297D/ A330Y/I332E 82 S239N/ A330L/I332E
30 N297D/I332E 83 S239N/ A330Y/I332E
31 N297D / I332E / S239D / A330L 84 S239N/I332D
32 N297D / S298A / A330Y / 1332E 85 S239N/I332E
33 N297D/T299L/I332E 86 S239N/I332N
34 N297D / T299F / I332E / N297D / 87 S239N/I332Q
T299H/I332E
35 N297D/T299I/I332E 88 S239N1S298A/I332E
36 N297D/T299L/I332E 89 S239Q/I332D
37 N297D/T299V/I332E 90 S239Q / I332E
38 N297E/I332E 91 S239Q / I332N
39 N297S / I332E 92 S239Q/I332Q
40 P230A/E233D/I332E 93 S239Q/ V264I/I332E
41 P244H / P245A / P247V 94 S298A/I332E
42 S239D/ A330L/I332E 95 V264E/N297D/I332E
43 S239D/ A330Y/I332E 96 V264I/ A330L/I332E
44 S239D / A330Y / I332E / K326E 97 V264I/ A330Y/I332E
45 S239D / A330Y / I332E / K326T 98 V264I/I332E
46 S239D / A330Y / I332E / L234I 99 V264I/ S298A/I332E
47 S239D / A330Y / I332E / L235D 100 Y296D/N297D/I332E
48 S239D / A330Y / I332E / V240I 101 Y296E/N297D/I332E
49 S239D / A330Y / I332E / V264T 102 Y296H/N297D/I332E
50 S239D / A330Y / I332E / V266I 103 Y296N/N297D/I332E
51 S239D / D265F / N297D / I332E 104 Y296Q/N297I/I332E
52 S239D / D265H / N297D / I332E 105 Y296T/N297D/I332E
53 S239D / D265I / N297D / I332E [0066] In particularly preferred embodiments, the invention encompasses B7-H3-binding molecules that comprise a variant Fc Domain wherein the variant confers or has an increased ADCC activity and/or an increased binding to FcyRIIIA (CD16A), and may also have a reduced binding to FcyRIIB (CD32B). Exemplary variants of human IgGl Fc Domains with increased binding to CD16A and which may additionally have reduced binding to CD32B contain L235V, F243L, R292P, Y300L, V305I or P296L substitutions. Preferred B7-H3- binding molecules include variant IgGl Fc Domains that include any 1, 2, 3, 4, 5, or 6 of the substitutions: L235V, F243L, R292P, Y300L, V305I and P396L. These amino acid substitutions may be present in a human IgGl Fc Domain in any combination.
[0067] In one embodiment, a B7-H3-binding molecule will comprise a variant Fc Domain having at least one modification in the Fc Domain. In certain embodiments, the variant Fc Domain comprises at least one substitution selected from the group consisting of L235V, F243L, R292P Y300L, V305L and P396L.
In a specific embodiment, the variant Fc Domain comprises:
at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L;
at least two substitutions selected from the group consisting of:
(1) F243L and P396L;
(2) F243L and R292P; and
(3) R292P and V305I;
at least three substitutions selected from the group consisting of:
(1) F243L, R292P and Y300L;
(2) F243L, R292P and V305I;
(3) F243L, R292P and P396L; and
(4) R292P, V305I and P396L;
at least four substitutions selected from the group consisting of:
(1) F243L, R292P, Y300L and P396L; and
(2) F243L, R292P, V305I and P396L; or
at least the five substitutions selected from the group consisting of:
(1) F243L, R292P, Y300L, V305I and P396L; and
(2) L235V, F243L, R292P, Y300L and P396L. [0069] In another specific embodiment, the variant Fc Domain comprises substitutions of:
(A) F243L, R292P, and Y300L;
(B) L235V, F243L, R292P, Y300L, and P396L; or
(C) F243L, R292P, Y300L, V305I, and P396L.
[0070] In certain embodiments the CTLA-4-binding molecule comprises a variant Fc Domain wherein the variant confers or has decreased (or substantially no) binding to FcyRIIIA (CD 16a), relative to the binding exhibited by the wild-type Fc Domain (SEQ ID NO:l)).
[0071] Exemplary variants of human IgGl Fc Domains with reduced binding to FcyRs contain L234A, L235A, D265A, N297A or N297Q, substitutions. Alternative CTLA-4- binding molecules useful in the invention include variant IgGl Fc Domains that include any 1, 2, 3, 4, or all 5, of the substitutions: L234A, L235A, D265A, N297A, and N297Q. These amino acid substitutions may be present in a human IgGl Fc Domain in any combination.
[0072] In one embodiment, a CTLA-4-binding molecule will comprise a variant Fc Domain having at least one modification in the Fc Domain. In certain embodiments, the variant Fc Domain comprises at least one substitution selected from the group consisting of L234A, L235A, D265A, and N297Q. Since the L234A, L235A, D265A, N297A, and N297Q substitutions abolish effector function, in circumstances in which effector function is desired, these substitutions would preferably not be employed.
[0073] In a specific embodiment, the variant Fc Domain comprises substitutions of:
(A) L234A, L235A;
(B) D265A;
(D) N297A; or
(C) N297Q.
[0074] A preferred IgGl sequence for the CH2 and CH3 Domains of a variant Fc Domain with reduced binding to FcyRs for use in the methods of the invention will have the
L234A/L235A substitutions (SEQ ID NO:5):
APEAAGGPSV FLFPPKPKDT LMI SRTPEVT CWVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNS TY RWSVLTVLH QDWLNGKEYK CKVSNKALPA
P IEKT I SKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGS FFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLS PGX wherein, X is a lysine (K) or is absent. [0075] In alternative embodiments, the CH2-CH3 Domain of an Fc Domain-containing CTLA-4-binding molecule for use in the methods of the present invention may be one that inherently exhibits decreased (or substantially no) binding to FcyRIIIA (CD 16a) and/or reduced effector function (relative to the binding exhibited by the wild-type IgGl Fc Domain (SEQ ID NO:l)). For example, the CH2-CH3 Domain of an Fc Domain-containing CTLA-4- binding molecule for use in the methods of the present invention may be an IgG2 Fc Domain or an IgG4 Fc Domain.
[0076] In an alternative embodiment, a CTLA-4-binding molecule for use in the methods of the present invention comprises an IgG4 Fc Domain. Where an IgG4 Fc Domain is utilized the instant invention also encompasses the introduction of a stabilizing hinge mutation such as S228P (e.g., ESKYGPPCPPCP (SEQ ID NO:14)), as numbered by the EU index as set forth in Kabat (Lu et al, (2008) "The Effect Of A Point Mutation On The Stability Of IgG4 As Monitored By Analytical Ultracentrifugation," J. Pharm. Sci. 97:960-969) to reduce the incidence of strand exchange. Other stabilizing mutations known in the art may be introduced into an IgG4 Fc Domain (Peters, P et al., (2012) "Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability," J. Biol. Chem., 287:24525-24533; PCT Patent Publication No: WO 2008/145142). Additionally, as noted above, when present, the CHI Domain and/or hinge is preferably of the same isotype as the desired Fc Domain. Accordingly, in such embodiments a CTLA-4-binding molecule (e.g. antibody) will comprise an IgG4 CHI (see, e.g., SEQ ID NO: 10), a stabilized IgG4 hinge (see, e.g., SEQ ID NO:47), and IgG4 CH2-CH3 Domains (see, e.g., SEQ ID NO:4),
[0077] The serum half-life of proteins comprising Fc Domains may be increased by increasing the binding affinity of the Fc Domain for FcRn. The term "half-life" as used herein means a pharmacokinetic property of a molecule that is a measure of the mean survival time of the molecules following their administration. Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known quantity of the molecule from the subject's body (e.g., human patient or other mammal) or from a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues. In general, an increase in half-life for an administered molecule results in an increase in that molecule's mean residence time (MRT) in circulation.
[0078] In some embodiments, the B7-H3-binding molecules and/or CTLA-4-binding molecules for use in the methods of the present invention comprise a variant Fc Domain, wherein the variant Fc Domain comprises at least one amino acid modification relative to a wild-type Fc Domain, such that the molecule has an increased half-life (relative to that of a wild-type Fc Domain).
[0079] In some embodiments, the B7-H3-binding molecules and/or CTLA-4-binding molecules for use in the methods of the present invention comprise a variant Fc Domain, wherein the variant Fc Domain comprises a half-live extending amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436. Numerous specific mutations capable of increasing the half-life of an Fc Domain-containing molecule are known in the art and include, for example M252Y, S254T, T256E, and combinations thereof. For example, see the mutations described in U.S. Patent Nos. 6,277,375; 7,083,784; 7,217,797; 8,088,376; U.S. Publication Nos. 2002/0147311; 2007/0148164; and International Publication Nos. WO 98/23289; WO 2009/058492; and WO 2010/033279, which are herein incorporated by reference in their entireties. Fc Domain-containing molecules with enhanced half-life also include those with substitutions at two or more of Fc Domain residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435 and 436. In particular, two or more substitutions selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, Y436I.
[0080] In a specific embodiment, the variant Fc Domain comprises substitutions of:
(A) M252Y, S254T and T256E;
(B) M252Y and S254T;
(C) M252Y and T256E;
(D) T250Q and M428L;
(E) T307Q and N434A;
(F) A378V and N434A;
(G) N434A and Y436I;
(H) V308P and N434A; or
(I) K288D and H435K.
The instant invention further encompasses variant Fc Domains comprising:
(A) one or more mutations which alter effector function and/or FcyR; and
(B) one or more mutations which extend serum half-life. [0082] The two CH2 and/or two CH3 Domains of the CH2-CH3 Domains of two interacting Fc Domain-containing polypeptide chains of an B7-H3-binding molecules and/or CTLA-4-binding molecule for use in the methods of the present invention need not be identical in sequence, and advantageously are modified to foster complexing between the two polypeptide chains (see, for example: WO 98/50431; WO2007/110205; WO2011/143545; WO 2012/058768; WO 2013/06867). For example, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a "knob", e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., "the hole" (e.g., a substitution with glycine). Such sets of mutations can be engineered into any of the polypeptides of the Fc Domain-containing B7-H3 -binding molecules and/or CTLA-4-binding molecules of the present invention. Methods of protein engineering to favor heterodimerization over homodimerization are well-known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996) " 'Knobs-Into-Holes ' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, " Protein Engr. 9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, " J. Mol. Biol. 270: 26-35, and Xie etal. (2005) "A New Format OfBi-specific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, " J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its entirety). Preferably the "knob" is engineered into the CH2-CH3 Domains of one polypeptide chain and the "hole" is engineered into the CH2-CH3 Domains of the other CH2- CH3 -containing polypeptide chain. Thus, the "knob" will help in preventing the first polypeptide chain from homodimerizing via its CH2 and/or CH3 Domains. The CH2-CH3 "hole-bearing" polypeptide chain will heterodimerize with the CH2-CH3 "knob-bearing" polypeptide chain, and will also homodimerize with itself. A preferred knob is created by modifying a native IgG Fc Domain to contain the modification T366W. A preferred hole is created by modifying a native IgG Fc Domain to contain the modification T366S, L368A and Y407V. To aid in purifying the "hole-bearing" polypeptide chain homodimer from the preferred heterodimer molecule, the protein A binding site of the CH2 and CH3 Domains of the "hole-bearing" Fc Domain is preferably mutated by amino acid substitution at position 435 (H435R). Thus, the "hole-bearing" Fc Domain homodimer will not bind to protein A, whereas an Fc Domain-containing B7-H3-binding molecule and/or CTLA-4-binding molecule for use in the methods of the present invention will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.
[0083] A preferred "knob-bearing" sequence for an Fc Domain-containing B7-H3- binding molecule and/or CTLA-4-binding molecule has the sequence (SEQ ID NO:6):
APELLGGPSV FL FPPKPKDT LMI SRT PEVT CWVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNS TY RWSVLTVLH QDWLNGKEYK CKVSNKALPA
P I EKT I SKAK GQPRE PQVYT LPPSREEMTK NQVS LWCLVK GFYPS D IAVE
WE SNGQPENN YKT T PPVLDS DGS FFLYSKL TVDKSRWQQG NVFS CSVMHE ALHNHYTQKS LS LS PGX wherein, X is a lysine (K) or is absent.
[0084] A preferred "hole-bearing" sequence for an Fc Domain-containing B7-H3-binding molecule and/or CTLA-4-binding molecule has the sequence (SEQ ID NO:7):
APELLGGPSV FL FPPKPKDT LMI SRT PEVT CWVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNS TY RWSVLTVLH QDWLNGKEYK CKVSNKALPA
P I EKT I SKAK GQPRE PQVYT LPPSREEMTK NQVS LSCAVK GFYPS D IAVE
WE SNGQPENN YKT T PPVLDS DGS FFLVSKL TVDKSRWQQG NVFS CSVMHE ALHNRYTQKS LS LS PGX wherein, X is a lysine (K) or is absent.
[0085] The invention also encompasses such CH2-CH3 Domains, which comprise additional substitutions which modify effector function and/or FyR binding activity of the Fc Domain as provided above. The invention also encompasses such CH2-CH3 Domains, which further comprise one or more half-live extending amino acid substitutions. In particular, the invention encompasses such hole-bearing and such knob-bearing CH2-CH3 Domains which further comprise the M252Y/S254T/T256E substitutions.
B. B7-H3-Binding Molecules
[0086] Molecules that specifically bind B7-H3 encompassed by the invention include anti- B7-H3 antibodies capable of binding to a continuous or discontinuous (e.g., conformational) portion (epitope) of human B7-H3, and molecules comprising the epitope-binding site of such antibodies. The B7-H3-binding molecules used in the methods and compositions of the present invention will preferably also exhibit the ability to bind to the B7-H3 molecules of one or more non-human species, especially, murine, rodent, canine, and primate species. Antibodies that are specific for B7-H3 are known (see, e.g., United States Patent Nos. 7,527,969; 7,666,424; 7,718,774; 7,737,258; 7,740,845; 8,148, 154; 8,216,570; 8,414,892; 8,501,471; 8,779,098; 8,802,091; 9,062,110; US Patent Publication Nos. 2013/0078234; 2010/0143245; and PCT Patent Publications WO 2004/001381; WO 2008/066691; WO 2008/116219; WO 2011/109400; WO 2012/147713, and Table 5). Additional desired antibodies may be made by isolating antibody-secreting hybndomas elicited using B7-H3 expressing cells; B7-H3 or a peptide fragment thereof.
[0087] Human B7-H3 exists as a "2Ig" form and as a "4Ig" form. The amino acid sequence of the "2Ig" form of human B7-H3 (including a 29 amino acid residue signal sequence, shown underlined) is (SEQ ID NO: 18):
MLRRRGS PGM GVHVGAALGA LWFCLTGALE VQVPEDPWA LVGTDATLCC
SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL
AQGNASLRLQ RVRVADEGSF TCFVS IRDFG SAAVSLQVAA PYSKPSMTLE
PNKDLRPGDT VTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG
LFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQ PMTFPPEALW
VTVGLSVCLI ALLVALAFVC WRKIKQSCEE ENAGAEDQDG EGEGSKTALQ PLKHSDSKED DGQEIA
[0088] The amino acid sequence of the "2Ig" form of human B7-H3 (SEQ ID NO: 18) is completely embraced within the "4Ig" form of human B7-H3(SEQ ID NO: 19, the 29 amino acid residue signal sequence, shown underlined):
MLRRRGS PGM GVHVGAALGA LWFCLTGALE VQVPEDPWA LVGTDATLCC
SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL
AQGNASLRLQ RVRVADEGSF TCFVS IRDFG SAAVSLQVAA PYSKPSMTLE
PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG
LFDVHS ILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ
VPEDPWALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG
RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVS IRDFGSA
AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ
GVPLTGNVTT SQMANEQGLF DVHSVLRWL GANGTYSCLV RNPVLQQDAH
GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN
AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA
[0089] Preferred anti-B7-H3-binding molecules possess the VL and/or VH Domains, of the anti-human B7-H3 monoclonal antibody "BRCA84D," "BRCA69D," "PRCA1," or any of the anti-B7-H3 antibodies provided in Table 5; and more preferably possess 1, 2 or all 3 of the CDRLS of the VL Region and/or 1, 2 or all 3 of the CDRHS of the VH Domain of such anti- B7-H3 monoclonal antibodies. Particularly preferred, are B7-H3-binding molecules which possess a humanized VH and/or VL Domain. Such preferred B7-H3-binding molecules include antibodies having variant Fc Domains, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, etc. 1. BRCA84D
[0090] The amino acid sequence of the VL Domain of BRCA84D (SEQ ID NO:20) is shown below (CDRL residues are shown underlined).
DIAMTQSQKF MS TSVGDRVS VTCKASQNVD TNVAWYQQKP GQS PKAL IYS ASYRYSGVPD RFTGSGSGTD FTLT INNVQS EDLAEYFCQQ YNNYPFTFGS
GTKLE IK
[0091] The amino acid sequence of the VH Domain of BRCA84D (SEQ ID NO:21) is shown below (CDRH residues are shown underlined).
DVQLVESGGG LVQPGGSRKL SCAASGFT FS SFGMHWVRQA PEKGLEWVAY ISSDSSAIYY ADTVKGRFT I SRDNPKNTLF LQMTSLRSED TAMYYCGRGR ENIYYGSRLD YWGQGTTLTV S S a. hBRCA84D
[0092] Six exemplary humanized VL Domains of BRCA84D designated herein as "hBRCA84D VLl," "hBRCA84D VL2," "hBRCA84D VL3," "hBRCA84D VL4," "hBRCA84D VL5," "hBRCA84D VL6," and four exemplary humanized VH Domains of BRCA84D designated herein as "hBRCA84D VH1," "hBRCA84D VH2," "hBRCA84D VH3," and "hBRCA84D VH4," are provided below. Any of the humanized VL Domains may be paired with any of the humanized VH Domains to generate a B7-H3 binding domain. Accordingly, any antibody comprising one of the humanized VL Domains paired with the humanized VH Domain is referred to generically as "hBRCA84D," and particular combinations of humanized VH/VL Domains are referred to by reference to the specific VH/VL Domains, for example a humanized antibody comprising hBRCA84D VHl and hBRCA84D VL2 is specifically referred to as "hBRCA84D (1.2)."
[0093] The amino acid sequence of the VL Domain of hBRCA84D VLl (SEQ ID NO:22) is shown below (CDRL residues are shown underlined).
DI QLTQS PS F LSASVGDRVT I TCKASQNVD TNVAWYQQKP GKAPKLL IYS ASYRYSGVPS RFSGSGSGTD FTLT I S SLQP EDFATYYCQQ YNNYPFTFGQ
GTKLE IK
[0094] The amino acid sequence of the VL Domain of hBRCA84D VL2 (SEQ ID NO:23) is shown below (CDRL residues are shown underlined).
DI QLTQS PS F LSASVGDRVT I TCKASQNVD TNVAWYQQKP GKAPKAL IYS ASYRYSGVPS RFSGSGSGTD FTLT I S SLQP EDFATYYCQQ YNNYPFTFGQ
GTKLE IK [0095] The amino acid sequence of the VL Domain of hBRCA84D VL3 (SEQ ID NO:24) is shown below (CDRL residues are shown underlined).
DIQLTQSPSF LSASVGDRVS VTCKASQNVD TNVAWYQQKP GKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ
GTKLEIK
[0096] The amino acid sequence of the VL Domain of hBRCA84D VL4 (SEQ ID NO:25) is shown below (CDRL residues are shown underlined).
DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GQAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ
GTKLEIK
[0097] The amino acid sequence of the VL Domain of hBRCA84D VL5 (SEQ ID NO:26) is shown below (CDRL residues are shown underlined).
DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GQAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ
GTKLEIK
[0098] The amino acid sequence of the VL Domain of hBRCA84D VL6 (SEQ ID NO:27) is shown below (CDRL residues are shown underlined).
DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYYCQQ YNNYPFTFGQ
GTKLEIK
[0099] The amino acid sequence of the VH Domain of hBRC A84D VH 1 (SEQ ID NO : 28) is shown below (CDRH residues are shown underlined).
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARGR ENIYYGSRLD YWGQGTTVTV SS
[00100] The amino acid sequence of the VH Domain of hBRC A84D VH2 (SEQ ID NO : 29) is shown below (CDRH residues are shown underlined).
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCGRGR ENIYYGSRLD YWGQGTTVTV SS
[00101] The amino acid sequence of the VH Domain of hBRCA84D VH3 (SEQ ID NO:30) is shown below (CDRH residues are shown underlined).
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRDED TAMYYCGRGR ENIYYGSRLD YWGQGTTVTV SS [00102] The amino acid sequence of the VH Domain of hBRCA84D VH4 (SEQ ID NO:31) is shown below (CDRH residues are shown underlined).
EVQLVESGGG LVQPGGSLRL SCAASGFT FS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRSED TAVYYCARGR ENIYYGSRLD YWGQGTTVTV S S
2. BRCA69D
[00103] The amino acid sequence of the VL Domain of BRCA69D (SEQ ID NO:32) is shown below (CDRL residues are shown underlined).
DI QMTQTTS S LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLL IYY TSRLHSGVPS RFSGSGSGTD YSLT I DNLEQ EDIATYFCQQ GNTLPPTFGG
GTKLE IK
[00104] The amino acid sequence of the VH Domain of BRCA69D (SEQ ID NO:33) is shown below (CDRH residues are shown underlined).
QVQLQQSGAE LARPGASVKL SCKASGYT FT SYWMQWVKQR PGQGLEWI GT IYPGDGDTRY TQKFKGKATL TADKS S S TAY MQLS SLASED SAVYYCARRG IPRLWYFDVW GAGTTVTVS S a. hBRCA69D
[00105] Two exemplary humanized VL Domains of BRCA69D designated herein as "hBRCA69D VL1," and "hBRCA69D VL2," and two exemplary humanized VH Domains of BRCA69D designated herein as "hBRCA69D VHl," and "hBRCA69D VH2," are provided below. It will be noted that hBRCA69D VL2 includes amino acid substitutions in CDRLI and CDRL2, and that hBRCA69D VH2 includes amino acid substitutions in CDRL2. Any of the humanized VL Domains may be paired with any of the humanized VH Domains to generate a B7-H3 binding domain. Accordingly, any antibody comprising one of the humanized VL Domains paired with the humanized VH Domain is referred to genetically as "hBRCA69D," and particular combinations of humanized VH/VL Domains are referred to by reference to the specific VH/VL Domains, for example a humanized antibody comprising hBRCA69D VHl and hBRCA69D VL2 is specifically referred to as "hBRCA69D (1.2)."
[00106] The amino acid sequence of the VL Domain of hBRCA69D VLl (SEQ ID NO:34) is shown below (CDRL residues are shown underlined).
DI QMTQS PS S LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLL IYY TSRLHSGVPS RFSGSGSGTD FTLT I S SLQP EDIATYYCQQ GNTLPPTFGG
GTKLE IK [00107] The amino acid sequence of the VL Domain of hBRCA69D VL2 (SEQ ID NO:35) is shown below (CDRL residues are shown underlined).
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYY TSRLQSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG
GTKLEIK
[00108] The amino acid sequence of the VH Domain of hBRCA69D VH1 is (SEQ ID NO:36) (CDRH residues are shown underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQWVRQA PGQGLEWMGT IYPGDGDTRY TQKFKGRVT I TADKSTSTAY MELSSLRSED TAVYYCARRG IPRLWYFDVW GQGTTVTVSS
[00109] The amino acid sequence of the VH Domain of hBRCA69D VH2 is (SEQ ID NO:37) (CDRH residues are shown underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQWVRQA PGQGLEWMGT IYPGGGDTRY TQKFQGRVT I TADKSTSTAY MELSSLRSED TAVYYCARRG IPRLWYFDVW GQGTTVTVSS
3. PRCA157
[00110] The amino acid sequence of the VL Domain of PRCA157 (SEQ ID NO:38) is shown below (CDRH residues are shown underlined).
DIQMTQSPAS LSVSVGETVT ITCRASESIY SYLAWYQQKQ GKSPQLLVYN TKTLPE GVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYCQH HYGTPPWTFG
GGTNLEIK
[00111] The amino acid sequence of the VH Domain of PRCA157 (SEQ ID NO:39) is shown below (CDRH residues are shown underlined).
EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT PDKRLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNTLY LQMRSLKSED TAMYYCARHD GGAMDYWGQG TSVTVSS
4. Additional Anti-B7-H3 Antibodies
[00112] Additional anti-B7-H3 antibodies which may be utilized in the methods and compositions of the instant invention are provided in Table 5.
Figure imgf000041_0001
5. Exemplary Anti-B7-H3 Antibodies
[00113] In certain embodiments B7-H3 antibodies useful in the methods and compositions of the instant inventions comprise the VL and VH Domains of any of the antibodies provided above (e.g., hBRCA84D, hBRCA69D, PRCA157, or the VL and VH Domains of any of the anti-B7-H3 antibodies in Table 5), a kappa CL Domain, and a variant IgGl Fc Domain having enhanced ADCC (relative to a wild-type Fc Domain). In one embodiment, the CH2-CH3 Domains comprise the L235V, F243L, R292P, Y300L and P396L substitutions (wherein the numbering is according to the EU index as in Kabat). Such antibodies will preferably comprise an IgGl CHI Domain and Hinge Domain.
[00114] The amino acid sequence of a kappa CL Domain (SEQ ID NO: 15) is shown below.
RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDS TYSLS S TLTLSKADYE KHKVYACEVT HQGLS S PVTK S FNRGEC
[00115] The amino acid sequence of an IgGl CHI Domain and Hinge (SEQ ID NO: 16) is shown below.
AS TKGPSVFP LAPS SKS TSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HT FPAVLQS S GLYSLS SWT VPS S SLGTQT YI CNVNHKPS NTKVDKRVEP KSCDKTHTCP PCP [00116] The amino acid sequence of IgGl CH2-CH3 Domains comprising L235V, F243L,
R292P, Y300L and P396L substitutions (SEQ ID NO:17) is shown below.
APELVGGPSV FLLPPKPKDT LMI SRTPEVT CWVDVSHED PEVKFNWYVD
GVEVHNAKTK PPEEQYNS TL RWSVLTVLH QDWLNGKEYK CKVSNKALPA
P IEKT I SKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPLVLDS DGS FFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLS PGK
[00117] An exemplary anti-B7-H3 antibody, designated "hBRCA84D-2," comprises: a light chain having the VL Domain of BRCA84D VL2 (SEQ ID NO:23) and a kappa CL (SEQ ID NO: 15); and a heavy chain having the VH Domain of BRCA84D VH2 (SEQ ID NO:29), an IgGl CHI Domain and Hinge (SEQ ID NO: 16), and variant IgG CH2-CH3 Domains comprising L235V, F243L, R292P, Y300L and P396L substitutions (SEQ ID NO: 17).
[00118] The amino acid sequence of the complete light chain of hBRCA84D-2 (SEQ ID
NO:40) is shown below.
DI QLTQS PS F LSASVGDRVT I TCKASQNVD TNVAWYQQKP GKAPKAL IYS
ASYRYSGVPS RFSGSGSGTD FTLT I S SLQP EDFATYYCQQ YNNYPFT FGQ
GTKLE IKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV
DNALQSGNSQ ESVTEQDSKD S TYSLS S TLT LSKADYEKHK VYACEVTHQG LS S PVTKS FN RGEC
[00119] The amino acid sequence of the complete heavy chain of hBRCA84D-2 (SEQ ID
NO:41) is shown below.
EVQLVESGGG LVQPGGSLRL SCAASGFT FS S FGMHWVRQA PGKGLEWVAY I S SDS SAIYY ADTVKGRFT I SRDNAKNSLY LQMNSLRDED TAVYYCGRGR ENI YYGSRLD YWGQGTTVTV S SAS TKGPSV FPLAPS SKS T SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHT FPAVLQ S SGLYSLS SV VTVPS S SLGT QTYI CNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELV GGPSVFLLPP KPKDTLMI SR TPEVTCWVD VSHEDPEVKF NWYVDGVEVH NAKTKPPEEQ YNS TLRWSV LTVLHQDWLN GKEYKCKVSN KALPAP IEKT I SKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP LVLDSDGS FF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLS P GK
C. CTLA-4-Binding Molecules
[00120] Molecules that specifically bind CTLA-4 encompassed by the invention include anti-CTLA-4 antibodies capable of binding to a continuous or discontinuous (e.g., conformational) portion (epitope) of human CTLA-4, and molecules comprising the epitope- binding site of such antibodies. The CTLA-4-binding molecules used in the methods and compositions of the present invention will preferably also exhibit the ability to bind to the CTLA-4 molecules of one or more non-human species, especially, murine, rodent, canine, and primate species. Antibodies that are specific for CTLA-4 are known (see, e.g., United States Patents 6,984,720; 6,682,736; 7,034,121; 7,109,003; 7,132,281; 7,411,057; 7,605,238; 7,807,797; 7,824,679; 8,017,114; 8,143,379; 8,318,916; 8,491,895; 8,784,815; 8,883,984; US Patent Publications 2009/0123477; 2009/0252741; 2014/0105914; and PCT Patent Publications WO 00/37504; WO 01/14424; WO 01/54732; WO 2006/029219; WO 2006/066568; WO 2012/120125; and Table 6). Additional desired antibodies may be made by isolating antibody-secreting hybridomas elicited using CTLA-4 or a peptide fragment thereof.
[00121] Human CTLA-4 (including a 35 amino acid residue signal sequence (shown underlined) and the 188 amino acid residue mature protein) has the amino acid sequence (SEQ ID NO:42):
MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQ PAWL ASS RGIASFVCEY ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN
[00122] Preferred anti-CTLA-4-binding molecules useful in the methods and compositions of the instant invention possess the VL and/or VH Domains of the anti-human CTLA-4 monoclonal antibody "CTLA-4 mAb 1" (ipilimumab, CAS Reg. No.:477202-00-9, also known as MDX010, and marketed as Yervoy® by Bristol-Myers Squibb); "CTLA-4 mAb 2" (tremelimumab, CAS Reg. No.: 745013-59-6, also known as CP-675206); or the VL and/or VH Domains of any of the anti-CTLA-4 antibodies in Table 6; and more preferably possess 1, 2 or all 3 of the CDRLS of the VL Region and/or 1, 2 or all 3 of the CDRHS of the VH Domain of such anti-CTLA-4 monoclonal antibodies. Such preferred anti-CTLA-4-binding molecules include antibodies having wild-type Fc Domains, variant Fc Domains, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, etc.
1. CTLA-4 mAb 1
[00123] The amino acid sequence of the VH Domain of CTLA-4 mAb 1 (SEQ ID NO:43) is shown below (CDRH residues are shown underlined).
QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAI YYCARTG WLGPFDYWGQ GTLVTVSS [00124] The amino acid sequence of the VL Domain of CTLA-4 mAb 1 (SEQ ID NO:44) is shown below (CDRL residues are shown underlined).
E IVLTQS PGT LSLS PGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLL IY GAFSRATGIP DRFSGSGSGT DFTLT I SRLE PEDFAVYYCQ QYGSSPWTFG QGTKVE IK
2. CTLA-4 mAb 2
[00125] The amino acid sequence of the VH Domain of CTLA-4 mAb 2 (SEQ ID NO:45) is shown below (CDRH residues are shown underlined).
QVQLVESGGG WQPGRSLRL SCAASGFT FS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP RGATLYYYYY GMDVWGQGTT VTVS S
[00126] The amino acid sequence of the VL Domain of CTLA-4 mAb 2 (SEQ ID NO:46) is shown below (CDRL residues are shown underlined).
DI QMTQS PS S LSASVGDRVT ITCRASQSIN SYLDWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLT I S SLQP EDFATYYCQQ YYSTPFTFGP
GTKVE IK
3. Additional anti-CTLA-4 Antibodies
[00127] Additional anti-CTLA-4 antibodies that may be utilized in the methods and compositions of the instant invention are provided in Table 6.
Figure imgf000044_0001
4. Exemplary CTLA-4 Antibodies
[00128] In certain embodiments CTLA-4 antibodies useful in the methods and compositions of the instant inventions comprise the VL and VH Domains of any of the antibodies provided above (e.g., CTLA-4 mAb 1, CTLA-4 mAb 2 or any of the anti-CTLA-4 antibodies in Table 6), a kappa CL Domain, and an IgGl, IgG2 or IgG4 Fc Domain, optionally lacking the C- terminal lysine residue. Such antibodies will preferably comprise a CHI Domain and a Hinge Domain of the same isotype as the Fc Domain.
[00129] An exemplary anti-CTLA antibody is ipilimumab (CAS Reg. No.:477202-00-9), which is a human antibody comprising a light chain having the VL Domain of CTLA-4 mAb 1 (SEQ ID NO:44), and a kappa CL Domain (see, e.g., SEQ ID NO:15); and a heavy chain have the VH Domain of CTLA-4 mAb 1 (SEQ ID NO:43), an IgGl CHI Domain (see, e.g., SEQ ID NO:8), an IgG Hinge (see, e.g., SEQ ID NO:ll), and IgGl CH2-CH3 Domains (see, e.g., SEQ ID NO:l)
[00130] Another exemplary anti-CTLA antibody is tremelimumab (CAS Reg. No. : 745013- 59-6), which is a human antibody comprising a light chain having the VL Domain of CTLA-4 mAb 2 (SEQ ID NO:46), and a kappa CL Domain (see, e.g., SEQ ID NO:15); and a heavy chain having the VH Domain of CTLA-4 mAb 12 (SEQ ID NO: 45), an IgG2 CHI Domain (see, e.g., SEQ ID NO:9), an IgG2 Hinge (see, e.g., SEQ ID NO:12), and IgG2 CH2-CH3 Domains (see, e.g., SEQ ID NO:2).
D. Methods of Production
[00131] B7-H3-binding molecules and CTLA-4-binding molecules encompassed by the present invention can be produced by methods known in the art, for example, synthetically or recombinantly (see, e.g., Kelley, R. F. et al. (1990) In: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, J.K. Ed., Plenum Press, N.Y., vol. 12, pp 1-19; Stewart, J.M et al. (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL; see also United States Patents Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925; Merrifield, B. (1986) "Solid Phase Synthesis " Science 232(4748):341-347; Houghten, R.A. (1985) "General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen- Antibody Interaction At The Level Of Individual Amino Acids," Proc. Natl. Acad. Sci. (U.S.A.) 82(15):5131-5135; Ganesan, A. (2006) "Solid-Phase Synthesis In The Twenty-First Century," Mini Rev. Med. Chem. 6(1):3-10). [00132] Alternatively, suitable B7-H3-binding molecules and/or CTLA-4-binding molecules having one or more of the CDRs of a desired anti-B7-H3 antibody and/or anti- CTLA-4 antibody may be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XENOMOUSE™ (Abgenix, Inc., Fremont, CA) and HUMAB -MOUSE® and TC MOUSE™ (both from Medarex, Inc., Princeton, NJ).
[00133] In a further alternative method, such binding molecules may be made recombinantly and expressed using any method known in the art. Antibodies may be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that may be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been disclosed (see, for example, Peeters et al. (2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19:2756; Lonberg, N. et al. (1995) "Human Antibodies From Transgenic Mice," Int. Rev. Immunol 13 :65-93; and Pollock et al. (1999) "Transgenic Milk As A Method For The Production Of Recombinant Antibodies," J. Immunol Methods 231 : 147-157). Suitable methods for making derivatives of antibodies, e.g. , humanized antibodies, bispecific antibodies, single-chain, etc. are known in the art. In another alternative, antibodies may be made recombinantly by phage display technology (see, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994) "Making Antibodies By Phage Display Technology," Annu. Rev. Immunol. 12.433- 455).
[00134] Vectors containing polynucleotides encoding polypeptides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.
[00135] Any host cell capable of overexpressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non- limiting examples of suitable mammalian host cells include but are not limited to COS, HeLa, and CHO cells. Preferably, the host cells express the cDNAs at a level of about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening the host cells for immunospecific binding to a cDNA expressed target (e.g., B7-H3 or CTLA-4) may be accomplished using an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.
[00136] The invention includes polypeptides comprising an amino acid sequence (preferably the epitope binding domain) of an anti-B7-H3 antibody and/or anti-CTLA-4- antibody provided herein. The polypeptides of this invention can be made by procedures known in the art. The polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis. Polypeptides, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis.
[00137] The invention includes modifications of the polypeptides of any such B7-H3- binding molecules and/or CTLA-4-binding molecules that do not significantly affect the properties of such molecules as well as variants that have enhanced or decreased activity. The modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs. Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar chemical properties as that of the original amino acid. Such conservative substitutions are known in the art, and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the Variable Domain. Changes in the Variable Domain can alter binding affinity and/or immunospecificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as the attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art.
[00138] The invention encompasses fusion proteins comprising one or more of the antibodies of this invention. In one embodiment, a fusion polypeptide is provided that comprises a light chain, a heavy chain or both a light and heavy chain. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains a VL Domain and a VH Domain of an antibody provided herein or produced from a publicly-deposited hybridoma. For purposes of this invention, an antibody fusion protein contains one or more epitope-binding sites that immunospecifically bind to B7-H3 and/or CTLA-4, and one or more polypeptide domains that immunospecifically bind to another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region.
E. Pharmaceutical Compositions
[00139] The present invention encompasses compositions comprising a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules. The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules, and a pharmaceutically acceptable carrier. In a preferred aspect, such compositions are substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side effects).
[00140] Where more than one therapeutic agent is to be administered, the agents may be formulated together in the same formulation or may be formulated into separate compositions. Accordingly, in some embodiments, the B7-H3 -binding molecule and the CTLA-4-binding molecule are formulated together in the same pharmaceutical composition. In alternative embodiments, the molecules are formulated in separate pharmaceutical compositions.
[00141] Various formulations of a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules, may be used for administration. In addition to the pharmacologically active agent(s), the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that are well known in the art and are relatively inert substances that facilitate administration of a pharmacologically effective substance or which facilitate processing of the active compounds into preparations that can be used pharmaceutically for delivery to the site of action. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, buffers, and skin penetration enhancers.
[00142] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Aqueous carriers, such as saline solutions, aqueous dextrose and glycerol solutions are preferred when the pharmaceutical composition is administered intravenously. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain a minor amount of a wetting or emulsifying agent, or a pH buffering agent. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
[00143] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided, so that the ingredients may be mixed prior to administration.
[00144] The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[00145] Preferably, the therapeutic agent {i.e., a B7-H3-binding molecule, a CTLA-4- binding molecule, or a combination of such molecules) is supplied as a dry sterile lyophilized powder in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule(s). In one embodiment, the therapeutic agent is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. In an alternative embodiment, the therapeutic agent is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the therapeutic agent.
[00146] The lyophilized therapeutic agent {i.e., a B7-H3-binding molecule, a CTLA-4- binding molecule, or a combination of such molecules) should be stored at between 2°C and 8°C in the original container and the agent should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, the therapeutic agent is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule(s), fusion protein, or conjugated molecule. Preferably, such therapeutic agent when provided in liquid form is supplied in a hermetically sealed container.
[00147] The invention also provides a pharmaceutical pack or kit comprising one or more containers containing a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules alone or with other agents, preferably with a pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[00148] A kit can comprise a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules. The kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.
F. Methods of Use
[00149] As discussed above, molecules that specifically bind to B7-H3 and molecules that specifically bind to CTLA-4 may be used for therapeutic purposes in subjects with cancer or other diseases. Accordingly, the present invention provides methods of treating cancer, comprising administering to a subject in need thereof a B7-H3-binding molecule and a CTLA- 4-binding molecule. In particular, the present invention encompasses such methods wherein, the B7-H3-binding molecule comprises the epitope-binding site of an anti-B7-H3 antibody provided herein, and wherein the CTLA-4-binding molecule comprises the epitope-binding site of an anti-CTLA-4 antibody provided herein. In one embodiment the B7-H3-binding molecule is an antibody. In one embodiment, the CTLA-4-binding molecule is an antibody. In a further embodiment, both the B7-H3 -binding molecule and CTLA-4-binding molecule are antibodies.
[00150] In one embodiment, a B7-H3-binding molecule and a CTLA-4-binding molecule are administered concurrently. As used herein, such "concurrent" administration is intended to denote:
(A) the administration of a single pharmaceutical composition that contains both a B7-H3-binding molecule and a CTLA-4-binding molecule. Such molecules may be the same molecule (e.g., a bispecific antibody), or may be distinct (e.g., an anti-B7-H3 antibody, or antigen-binding fragment thereof, and an anti- CTLA-4-antibody, or antigen-binding fragment thereof); or
(B) the separate administration of two or more pharmaceutical compositions, one composition of which contains a molecule that specifically binds to B7-H3 and another composition of which contains a molecule that specifically binds to CTLA-4, wherein the compositions are administered within a 48-hour period.
[00151] In a second embodiment, two distinct molecules are employed, and the molecules are administered "sequentially" (e.g., an anti-B7-H3 antibody is administered and, at a later time, an anti-CTLA-4 antibody is provided, or vice versa). In such sequential administration, the second administered composition is administered at least 48 hours, or more after the administration of the first administered composition.
[00152] Providing a therapy or "treating" refers to any indicia of beneficial or desired results including, without limitation, clinical results such as shrinking the size of a tumor (in the cancer context, for example, a tumor of breast, gastric or prostate cancer), retardation of cancer cell growth, delaying the development of metastasis, decreasing a symptom resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and/or prolonging survival of an individual.
[00153] Subjects for treatment include animals, most preferably mammalian species such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as a cynomolgus monkey, human, etc.). In a preferred embodiment, the subject is a human.
[00154] Exemplary disorders that may be treated by various embodiments of the present invention include, but are not limited to, proliferative disorders, cell proliferative disorders, and cancer (especially a B7-H3 -expressing cancer). In various embodiments, the invention encompasses methods and compositions for treatment, prevention or management of a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount a molecule that specifically binds to B7-H3 and a molecule that specifically binds to CTLA-4. For example, the B7-H3-binding molecule and the CTLA-4-binding molecule are particularly useful for the prevention, inhibition, reduction of growth or regression of primary tumors, and metastasis of cancer cells. Although not intending to be bound by a particular mechanism of action, such binding molecules may mediate effector function against cancer cells, promote the activation of the immune system against cancer cells, cross-link cell-surface antigens and/or receptors on cancer cells and enhance apoptosis or negative growth regulatory signaling, or a combination thereof, resulting in tumor clearance and/or tumor reduction.
[00155] As used herein, an "effective amount" of a pharmaceutical composition, in one embodiment, is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as decreasing symptoms resulting from the disease attenuating a symptom of infection (e.g., viral load, fever, pain, sepsis, etc.) or a symptom of cancer (e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.), thereby increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and/or prolonging survival of individuals. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. Particular dosages are discussed below.
[00156] In one embodiment, a B7-H3-binding molecule (e.g., an antibody) and a CTLA-4- binding molecule (e.g., an antibody) can be used to treat any disease or condition associated with or characterized by the expression of B7-H3. Thus, without limitation, the methods and compositions of the instant invention may be used for immunotherapy directed at cancer including cancers characterized by the presence of a cancer cell, including but not limited to a cell of an acute myeloid leukemia, an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, a glioblastoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a malignant mesothelioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, a non-small cell lung cancer, an ovarian cancer, a pancreatic cancer, a pharyngeal cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterious uveal melanoma, a rare hematologic disorder, a renal cell carcinoma, a renal metastatic cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, a soft- tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, and a uterine cancer. Such immunotherapy may, for example, be sufficient to reduce cell division in the cancer cell, delay the development (e.g., onset and extent) of metastasis, and/or to promote the activity of the immune system on the cancer cells.
[00157] In particular, the combination of a B7-H3-binding molecule and a CTLA-4-binding molecule is particularly useful for the treatment of squamous cell cancers of the head and neck (SCCHN), bladder cancers, breast cancers, colorectal cancers, gastric cancers, glioblastomas, kidney cancers, lung cancers including non-small cell lung cancers (NSCLC), melanomas, ovarian cancers, pancreatic cancers, pharyngeal cancers, prostate cancers, renal cell carcinomas, and small round blue cell tumors of childhood including neuroblastomas and rhabdomyosarcomas, each of which highly express B7-H3.
[00158] It is understood that the B7-H3-binding molecules and CTLA-4-binding molecules are administered at a concentration that promotes binding at physiological (e.g., in vivo) conditions. The B7-H3-binding molecules and CTLA-4-binding molecules (e.g., anti-B7-H3 and anti-CTLA-4 antibodies) may be administered with additional agents that enhance or direct an individual's own immune response, such as an agent that strengthens ADCC or stimulated T-cells.
[00159] In yet another embodiment, the B7-H3-binding molecule and/or the CTLA-4- binding molecule may be conjugated to or associated with a radioactive molecule, toxin (e.g., calicheamicin), chemotherapeutic molecule, liposomes or other vesicles containing chemotherapeutic compounds and administered to an individual in need of such treatment to target these compounds to the cancer cell containing the antigen recognized by the B7-H3- binding molecule and thus eliminate cancer or diseased cells. Without being limited to any particular theory, the B7-H3-binding molecule (e.g., an anti-B7-H3 antibody) is internalized by the cell bearing B7-H3 at its surface, thus delivering the conjugated moiety to the cell to induce the therapeutic effect and the CTLA-4-binding molecule promotes the activation of the immune system.
[00160] In yet another embodiment, the B7-H3-binding molecule and CTLA-4-binding molecule (e.g., anti-B7-H3 and anti-CTLA-4 antibodies) can be employed as an adjuvant therapy at the time of the surgical removal of a tumor in order to delay, suppress or prevent the development of metastasis. The molecules can also be administered before surgery (neoadjuvant therapy) in order to decrease the size of the tumor and thus enable or simplify surgery, spare tissue during surgery, and/or decrease any resulting disfigurement.
[00161] Molecules having an Fc Domain with an increased affinity for FcyRIIIA and/or FcyRIIA, and optionally a decreased affinity for FcyRIIB, may lead to an enhanced activating response upon FcyR binding and thus have enhanced therapeutic efficacy for treating and/or preventing cancer. Accordingly, B7-H3-binding molecules comprising a variant Fc Domain are particularly useful for the treatment and/or prevention of a disease, or disorder where an effector cell function (e.g., ADCC) mediated by FcyR is desired (e.g., cancer). For example, a B7-H3 -binding molecule having enhanced FcyRIIIA binding may bind a cell-surface antigen and FcyRIIIA on an immune effector cell (e.g., K cell), stimulating an effector function (e.g., ADCC, CDC, phagocytosis, opsonization, etc.) against the cell. In some embodiments, the B7-H3-binding molecules provided herein are especially suited for the treatment of cancers. The efficacy of standard monoclonal antibody therapy depends on the FcyR polymorphism of the subject (Cartron, G. et al. (2002) "Therapeutic Activity Of Humanized Anti-CD20 Monoclonal Antibody And Polymorphism In IgG Fc Receptor FcgammaRIIIa Gene," Blood 99:754-758; Weng, W.K. et al. (2003) "Two Immunoglobulin G Fragment C Receptor Polymorphisms Independently Predict Response To Rituximab In Patients With Follicular Lymphoma," J Clin Oncol. 21(21):3940-3947). These receptors are expressed on the surface of the effector cells and mediate ADCC. High affinity alleles improve the effector cells' ability to mediate ADCC. In particular the B7-H3-binding molecules provided herein comprising a variant Fc Domain that exhibits enhanced affinity to FcyRIIIA (relative to a wild-type Fc Domain) on effector cells are better immunotherapy reagents for patients regardless of their FcyR polymorphism. G. Administration and Dosage
[00162] A combination of a B7-H3-binding molecule and a CTLA-4-binding molecule, may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with cancer or other disease, or disorder by administering to a subject an effective amount of such a combination, or pharmaceutical composition(s) comprising the same. In any of the embodiments below, the cancer is preferably a B7-H3 expressing cancer.
[00163] As used herein, the term "combination" refers to the use of more than one therapeutic agent (e.g., a B7-H3-binding molecule and a CTLA-4-binding molecule). The use of the term "combination" does not restrict the order in which therapeutic agents are administered to a subject with a disorder, nor does it mean that the agents are administered at exactly the same time, but rather it is meant that the agents are administered to a subject in a sequence and within a time interval such that the agents can act to provide an increased benefit than if they were administered otherwise. For example, each therapeutic agent (e.g., (i.e., a B7-H3-binding molecule and a CTLA-4-binding molecule) may be administered at the same time or sequentially in any order and/or at different points in time so as to provide the desired therapeutic or prophylactic effect. Furthermore, each agent need not be administered for the entire course of treatment. For example, both agents may be administered for a period of time, after which one agent is discontinued. Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route, e.g., one by the oral route and one parenterally.
[00164] A variety of delivery systems and administration routes for providing a combination of a B7-H3-binding molecule and a CTLA-4-binding molecule are available. Delivery systems that can be used to administer a B7-H3-binding molecule and a CTLA-4-binding molecule, include, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) "Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, " J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering a B7-H3-binding molecule and a CTLA-4-binding molecule, include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, molecules are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent (see, e.g., U.S. Patent Nos. 6,019,968; 5,985, 320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety).
[00165] Treatment of a subject with a therapeutically or prophylactically effective amount of a B7-H3-binding molecule and/or a CTLA-4-binding molecule can include a single treatment or, preferably, can include a series of treatments. For example, a subject may be treated with a B7-H3-binding molecule and/or a CTLA-4-binding molecule once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months for between about 2 to about 52 weeks. It will be appreciated that the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment. As provided herein, the B7-H3-binding molecule and the CTLA-4-binding molecule need not be administered at the same time or at the same intervals or for the same number of treatments.
[00166] Preferably the B7-H3-binding molecule and the CTLA-4-binding molecule are administered using a treatment regimen comprising one or more doses, wherein the treatment regimen is administered over 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 or more than 8 weeks. In certain embodiments, the treatment regimen comprises intermittently administering doses of the effective amount of such molecules (for example, administering a dose on week one and week four and not administering doses of the molecule on week two or week three). Typically, there are 1, 2, 3, 4, 5 or more than 5 courses of treatment. Each course may be the same regimen or a different regimen.
[00167] The dosage of a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules, administered to a patient is typically at least about at least about 1.0 mg/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, at least about 15 mg/kg body weight, or at least about 20 mg/kg. For antibodies encompassed by the invention, the dosage administered to a patient is typically 1.0 mg/kg to 20 mg/kg of the patient' s body weight. Preferably, the dosage administered to a patient is between 1.0 mg/kg and 20 mg/kg, 1.0 mg/kg and 10 mg/kg, 1.0 mg/kg and 5 mg/kg, 2.0 mg/kg and 20 mg/kg, or 5 mg/kg and 20 mg/kg of the patient' s body weight. In one embodiment, the dosage administered to a patient is between 1 mg/kg and 15 mg/kg body weight. In another embodiment, the dosage administered to a patient is 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg body weight. The calculated dose will be administered based on the patient' s body weight at baseline. Significant (> 10%) change in body weight from baseline or established plateau weight should prompt recalculation of dose.
[00168] Alternatively, a fixed dosage of a B7-H3-binding molecule, a CTLA-4-binding molecule, or a combination of such molecules is administered to a patient regardless of body weight. For antibodies encompassed by the invention, the fixed dosage administered to a patient is typically between 50 mg to 500 mg. Preferably, the fixed dosage administered to a patient is between 50 mg and 300 mg, 100 mg and 300 mg, or 100 mg and 200 mg. In one embodiment, the fixed dosage administered to a patient is 100 mg, 200 mg or 300 mg.
[00169] In various embodiments, a first therapeutic agent (e.g. , anti-B7-H3 antibody or anti- CTLA-4 antibody) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second (or subsequent) therapeutic agent (e.g., anti-B7-H3 antibody or anti-CTLA-4 antibody) to a subject with a disorder. In preferred embodiments, two or more agents are administered within the same patient visit.
[00170] As provided herein, the B7-H3-binding molecule and the CTLA-4-binding molecule may be administered at different dosages, different concentrations, different times and/or on different schedules.
[00171] Although, as discussed above, various dosing regimens and administration routes may be employed in order to provide a combination of a molecule that specifically binds to B7-H3 and a molecule that specifically binds to CTLA-4 to recipient subjects in need thereof in accordance with the present invention, certain combinations, dosing regimens and administrative routes are particularly preferred for use in such treatment. The use of an anti- B7-H3 antibody (e.g., hBRCA84D-2) alone and in combination with an anti-CTLA-4 antibody (e.g., ipilimumab) in such dosing and administrative is particularly preferred.
[00172] In certain embodiments, a dose of an anti-B7-H3 antibody is administered weekly in combination with a dose of an anti-CTLA-4 antibody administered every three or twelve weeks (wherein each administration of such a combination treatment regimen is herein referred to as a "cycle"). In one embodiment, 1 to 15 mg (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg), of an anti-B7-H3 antibody per kg of patient body weight is administered weekly, and 1 to 15 mg (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg), of an anti-CTLA-4 antibody per kg patient body weight is administered once every three weeks until remission of disease or unmanageable toxicity is observed. In preferred dosing regiments, treatment is divided into two periods, an "Induction Period" during which a dose of an anti-B7-H3 antibody is administered weekly in combination with a dose of an anti-CTLA-4 antibody administered every three weeks, followed by a "Maintenance Period" during which a dose of an anti-B7- H3 antibody is administered weekly alone (i.e., no further doses of an anti-CTLA-4 antibody are administered), or in combination with a dose of an anti-CTLA-4 antibody administered every twelve weeks.
[00173] In certain embodiments, an anti-B7-H3 antibody is administered to the subject by IV infusion weekly and an anti-CTLA-4 antibody is administered to the subject by IV infusion every three weeks for an Induction Period having a duration of at least 1 month or more, at least 3 months or more, or at least 6 months or more, or at least 12 months or more. In further embodiments, an anti-B7-H3 antibody is administered to the subject by IV infusion weekly alone, or in combination with an anti-CTLA-4 antibody administered to the subject by IV infusion every twelve weeks for a Maintenance Period having a duration of at least 1 month or more, at least 3 months or more, or at least 6 months or more, or at least 12 months or more. A total treatment duration of at least 3 months or more, or for least 6 months or more, or for at least 12 months or more, or until remission of disease or unmanageable toxicity is observed, is particularly preferred. In such IV administration once every three weeks (or twelve weeks during a Maintenance Period) an anti-B7-H3 antibody and an anti CTLA-4 antibody may be administered together or sequentially. In particularly preferred embodiments, an anti-B7-H3 antibody and an anti-CTLA-4 antibody are administered to the subject sequentially by IV infusion no more than 48 hours apart. In such sequential administration an anti-B7-H3 antibody may be administered prior to, or subsequent to the administration of an anti-CTLA-4 antibody.
[00174] It is particularly preferred to provide a subject with multiple doses of a combination treatment of an anti-B7-H3 antibody and an anti-CTLA-4 antibody. A treatment regimen may thus comprise, an Induction Period comprising 1 cycle, at least 2 cycles or more than 2 cycles, at least 3 cycles or more than 3 cycles, at least 4 cycles or more than 4 cycles, at least 5 cycles or more than 5 cycles, or at least 6 cycles or more than 6 cycles, and a an Maintenance Period comprising 1 cycle, at least 2 cycles or more than 2 cycles, at least 3 cycles or more than 3 cycles, at least 4 cycles or more than 4 cycles, at least 5 cycles or more than 5 cycles, or at least 6 cycles or more than 6 cycles . The dosage of each antibody in each such cycle may be the same or may vary from the prior administered dosage, the length of each cycle may be the same or may vary from the previous cycle, and each antibody need not be administered over the entire course of treatment.
[00175] It is preferred that the antibodies not be administered as an IV push or bolus, but rather that such administration be accomplished by IV infusion. The antibodies are thus preferably diluted into an infusion bag comprising a suitable diluent, e.g., 0.9% sodium chloride. Since infusion or allergic reactions may occur, premedication for the prevention of such infusion reactions is recommended and precautions for anaphylaxis should be observed during the antibody administration. It is particularly preferable for the IV infusion to be administered to the subject over a period of between 30 minutes and 24 hours. In certain embodiments, the IV infusion is preferably delivered over a period of 30-180 minutes, or 30- 120 minutes, or 30-90 minutes, or over a period of 60 minutes, or over a lesser period, if the subject does not exhibit signs or symptoms of an adverse infusion reaction.
[00176] Accordingly, a preferred method of treating cancer is provided, the method comprising administering to a subject in need thereof a combination of an anti-B7-H3 antibody and an anti-CTLA-4 antibody in an Induction Period, followed by a Maintenance Period, wherein:
(I) during the Induction Period, the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight {i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) every three weeks, such that a total of four doses of the anti-CTLA-4 antibody is administered and a total of no more than 10 doses of the anti-B7-H3 antibody is administered, and
(II) during the Maintenance Period, either:
(A) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, without any administration of the anti-CTLA-4 antibody; or
(B) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) once every twelve weeks, for the duration of such Maintenance Period.
[00177] In one embodiment, the dosage of the anti-B7-H3 antibody is 1, 3, 10, or 15 mg/kg body weight. In a further embodiment, the dosage of the anti-CTLA-4 antibody is 1, 3, 6, 10, or 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 1 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 3 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody is 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 10 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 1 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA- 4 antibody is 3 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 6 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 10 mg/kg body weight. In a further embodiment, the dosage of the anti-B7-H3 antibody is 15 mg/kg body weight and the dosage of the anti-CTLA-4 antibody is 15 mg/kg body weight. In any of the above embodiments, the anti-B7-H3 antibody and the anti-CTLA-4 antibody are administered by IV infusion, and when administered in the same week, both may be administered within a 48 hour, preferably 24-hour period.
[00178] In any of the above embodiments, the anti-B7-H3 antibody comprises the CDRLI CDRL2, CDRL3, CDRHI , CDRH2, and CDRH3 Domains of hBRCA84D, hBRCA69D, PRCA157, or the CDRL I CDRL2, CDRL3, CDRHI , CDRH2, and CDRH3 Domains of any of the anti-B7-H3 antibody provided in Table 5, and the anti-CTLA-4 antibody comprises the CDRLI CDRL2, CDRL3, CDRHI , CDRH2, and CDRH3 Domains of CTLA-4 mAb 1 , CTLA-4 mAb 2, or the CDRLI CDRL2, CDRL3, CDRH I, CDRH2, and CDRH3 Domains of any of the anti-CTLA-4 antibodies provided in Table 6. In any of the above embodiments, it is preferred that the anti-B7-H3 antibody be hBRCA84D-2 and that the anti-CTLA-4 antibody be selected from the antibodies provided in Table 6. In a preferred embodiment, the anti-B7-H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is ipilimumab. In another preferred embodiment, the anti-B7-H3 antibody is hBRCA84D-2 and the anti-CTLA-4 antibody is tremelimumab. [00179] As indicated above, the therapeutic agents are preferably cyclically administered to a subject. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improves the efficacy of the treatment. Exemplary cycles are about once every week, about once every 10 days, about once every two weeks, and about once every three weeks. Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest, etc. The number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.
[00180] A preferred dosage regimen for the therapeutic administration as provided in any of the embodiments above comprises administering an anti-B7-H3 antibody and an anti-CTLA-4 antibody to a recipient subject in an initial cycle, and one or more subsequent cycles. In the Induction Period of any of the embodiments provided above, the anti-B7-H3 antibody and the anti-CTLA-4 antibody are administered on the same cycle schedule. In such embodiments, each cycle preferably comprises three weeks, with the anti-B7-H3 antibody being provided to the subject in a weekly administration and the anti-CTLA-4 antibody being provided to the subject on the first week of each 3 -week period.
[00181] Alternatively, the anti-B7-H3 antibody and the anti-CTLA-4 antibody may be administered on different cycle schedules. In such embodiments, each cycle for the anti- CTLA-4 antibody will preferably comprise three weeks, with the anti-CTLA-4 antibody being provided to the subject on the first week of each 3 -week period. In such an embodiment, the initial cycle for the anti-B7-H3 antibody will preferably comprise 8 weeks, with the anti-B7- H3 antibody being provided to the subject in a weekly administration for the first four weeks of such 8-week initial period, followed by no administration to the subject for the period of weeks 5-8 of such 8-week initial period. Each subsequent cycle will preferably comprise a 4- week period, with the anti-B7-H3 antibody being provided to the subject in a weekly administration for the first three weeks of such 4-week subsequent period, followed by no administration to the subject for the period of week 4 of such 4-week subsequent period. Thus, for example, a subject would receive the B7-H3 antibody weekly in week 1, 2, 3, 4, 9, 10, 11, 13, 14, 15, etc., wherein weeks 1-8 are the initial dosage regimen cycle, weeks 9-12 are the first subsequent cycle, weeks 13-16 are a second subsequent cycle, etc. In the Induction Period of such embodiments, the subject would receive the CTLA-4 antibody every three weeks for a total of four doses {i.e., weeks 3, 6, 9 and 12). Accordingly: (I) during the Induction Period:
(A) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for 4 weeks (e.g., first administration cycle being weeks 1-4), followed by a 4-week suspension of such administration {e.g., in weeks 5-8), followed by a 2-week resumption of such administration {e.g., in weeks 9-10, such weeks being the first two weeks of the second administration cycle), and
(B) the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight {i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) every three weeks {e.g., in weeks 1, 4, 7 and 10), such that a total of four doses of the anti-CTLA-4 antibody is administered, and
(II) during the Maintenance Period, either:
(A) the anti-CTLA-4 antibody is not administered, and the anti-B7-H3 antibody is administered in one or more four week cycles at a dosage of 1 to 15 mg/kg body weight {i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for the first three weeks of a cycle, and with no administration of the anti-B7-H3 antibody in the fourth week of each cycle; or
(B) the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight {i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) once every twelve weeks, for the duration of such Maintenance Period, and the anti-B7-H3 antibody is administered in three or more cycles, each cycle being of four week duration, at a dosage of 1 to 15 mg/kg body weight {i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg body weight) weekly for the first three weeks of a cycle, and with no administration of the anti-B7-H3 antibody in the fourth week of each cycle.
I. Combination Therapies
[00182] The invention further encompasses administering to a subject, a combination of a molecule that specifically binds to B7-H3 and a molecule that specifically binds to CTLA-4 in further combination with other therapies known to those skilled in the art for the treatment or prevention of cancer, autoimmune disease, inflammation, or infectious disease, including but not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery. In some embodiments, the combination of a B7-H3-binding molecule and a CTLA-4-binding molecule {e.g., an anti- B7-H3 antibody and an anti-CTLA-4 antibody) are administered in combination with a therapeutically or prophylactically effective amount of one or more additional therapeutic agents known to those skilled in the art for the treatment and/or prevention of cancer, in particular a B7-H3 -expressing cancer.
[00183] In an embodiment for the treatment of a cell proliferative disorder, a B7-H3-binding molecule and/or a CTLA-4-binding molecule (e.g., anti-B7-H3 antibody, anti-CTLA-4 antibody) is conjugated to, or administered in further combination with, another therapeutic agent, such as, but not limited to, an alkylating agent (e.g., mechlorethamine or cisplatin), angiogenesis inhibitor, anthracycline (e.g., daunorubicin/daunomycin or doxorubicin), antibiotic (e.g., dactinomycin, bleomycin, or anthramycin), antibody (e.g., an anti-VEGF antibody such as bevacizumab (sold as AVASTIN® by Genentech, Inc.), an anti-EGFR antibody such as panitumumab (sold as VECTIBIX™ by Amgen, Inc.), or an anti-integrin antibody such as natalizumab (sold as TYSABRI® by Biogen Idee and Elan Pharmaceuticals, Inc.)), an antimetabolite (e.g., methotrexate or 5-fluorouracil), an anti -mitotic agent (e.g., vincristine or paclitaxel), a cytotoxin (e.g., a cytostatic or cytocidal agent), a hormone therapy agent (e.g., a selective estrogen receptor modulator (e.g., tamoxifen or raloxifene), aromatase inhibitor, luteinizing hormone-releasing hormone analog, progestational agent, adrenocorticosteroid, estrogen, androgen, anti-estrogen agent, androgen receptor blocking agent, 5-alpha reductase inhibitor, adrenal production inhibitor, etc.), a matrix metalloprotease inhibitor, a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.), or any other chemotherapeutic agent.
[00184] Non-limiting examples of suitable angiogenesis inhibitors include ABT-627; angiostatin (plasminogen fragment); angiozyme; anti angiogenic antithrombin III; Bay 12- 9566; benefin; bevacizumab; BMS-275291; bisphosphonates; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagen XVIII fragment); farnesyl transferase inhibitors (FTI); fibronectin fragment; gro- beta; halofuginone; heparinases; heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); EVI-862; interferon alpha/beta/gamma; interferon inducible protein (IP- 10); interleukin-12; kringle 5 (plasminogen fragment); marimastat; metalloproteinase inhibitors (TIMPs); 2-methoxy estradiol; MMI 270 (CGS 27023 A); MoAb FMC-1C11; neovastat; NM-3; panzem; PI-88; placental ribonuclease inhibitor; plasminogen activator inhibitor; platelet factor-4 (PF4); prinomastat; prolactin 16kDa fragment; proliferin-related protein (PRP); PTK 787/ZK 222594; retinoids; solimastat; squalamine; SS 3304; SU 5416; SU6668; SU11248; tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; thrombospondin-1 (TSP-1); TNP-470; transforming growth factor-beta (TGF-b); vasculostatin; vasostatin (calreticulin fragment); ZD6126; and ZD 6474.
[00185] Non-limiting examples of additional antibodies for the treatment of a cell proliferative disorder include antibodies to 17-1 A, ανβ3, AFP, CD3, CD18, CD20, CD22, CD33, CD44, CD52, CEA, CTLA-4, DNA-associated proteins, EGF receptor, Ep-CAM, GD2- ganglioside, gp Illb/IIIa, gp72, HLA-DR 10 beta, HLA-DR antigen, IgE, ganglioside GD3, MUC-1, nuC242, PEM antigen, SK-1 antigen, tumor antigen CA125, tumor antigen MUCl, VEGF, and VEGF-receptor.
Examples
[00186] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.
Example 1
Dose-Escalation Study of a Combination of an Anti-B7-H3 Antibody and an Anti-
CTLA-4 Antibody
[00187] While following dose-escalation study protocol details the use of the exemplary anti-B7-H3 antibody "hBRCA84D-2" in combination with the anti-CTLA-4 antibody "ipilimumab" it will be understood in view of the teachings herein that similar combination protocols may be designed using any of anti-B7-H3 antibodies and anti-CTLA-4 antibodies provided herein.
[00188] A dose escalation study is performed to determine the Maximum Tolerated Dose (MTD) or Maximum Administered Dose (MAD) (if no MTD is defined) of escalating doses of hBRCA84D-2 administered in combination with a dose of 3 mg/kg ipilimumab. This may be followed by a cohort expansion phase to further define the safety and initial efficacy of the combination with the hBRCA84D-2 dose established in the dose escalation study.
[00189] The dosing portion of the study comprises two distinct periods consisting of: 1) an Induction Period, and 2) a Maintenance Period. Figure 1 provides a schematic of the schedule of administration during the Induction and Maintenance Periods. [00190] The Induction Period constitutes the time when hBRCA84D-2 is administered in combination with ipilimumab. During the Induction Period hBRCA84D-2 is administered weekly and ipilimumab is administered once every 3 weeks. Once every three weeks the agents are administered on the same day with ipilimumab administered first, followed by hBRCA84D- 2. However, where the combined dose exceeds 2,500 mg the antibodies should be administered on consecutive days. When administering the antibodies on consecutive days ipilimumab may be administered on the first day, and hBRCA84D-2 may be administered on the next calendar day. Each cycle of therapy is defined as 3 weeks, in which hBRCA84D-2 is given on Days 1, 8 and 15, and ipilimumab is given on Day 1. The Induction Periods ends after a total of four doses of ipilimumab have been administered.
[00191] The Maintenance Period begins after the completion of the Induction period and constitutes the time when hBRCA84D-2 is administered without additional doses of ipilimumab. During the Maintenance Period hBRCA84D-2 is administered weekly.
[00192] hBRCA84D-2 may be evaluated in three sequential escalating doses, 3 mg/kg body weight, 10 mg/kg, and 15 mg/kg body weight, in combination with 3 mg/kg ipilimumab in cohort's patients. If it is determined that the MTD is exceeded in the first dose cohort, a dose de-escalation cohort to evaluate a lower dose of ipilimumab (1 mg/kg) in combination with 3, 10, or 15 mg/kg hBRCA84D-2 ipilimumab may be utilized.
[00193] Tumor assessments may be performed during the study, preferably at weeks 9, 18, 27, and every twelve weeks thereafter (i.e., weeks 39 and 51).
[00194] For a cohort expansion phase additional patients are enrolled and will receive hBRCA84D-2 at the MTD (or MAD) established from the dose escalation phase of the study in combination with 1 or 3 mg/kg ipilimumab.
[00195] Patients who remain clinically stable and do not experience unacceptable toxicity that necessitates permanent discontinuation of the study drugs, at the completion of the Induction Period will be eligible to participate in the Maintenance Phase and receive additional treatment with hBRCA84D-2. Patients that remain clinically stable, maintain a response status of stable disease or better, and do not experience unacceptable toxicity that necessitates permanent discontinuation of the study drugs, patients may receive additional treatment cycles of hBRAC84D-2. Such additional treatment may continue for -one year such that patients may receive 51 doses of hBRCA84D-2. [00196] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What Is Claimed Is:
Claim 1. A method of treating cancer, comprising administering to a subject in need thereof:
(a) a molecule that specifically binds to B7-H3, and
(b) a molecule that specifically binds to CTLA-4.
Claim 2. The method of claim 1, wherein said molecule that specifically binds to B7-H3 is an anti-B7-H3 antibody or antigen-binding fragment thereof, and said molecule that specifically binds to CTLA-4 is an anti-CTLA-4 antibody, or an antigen-binding fragment thereof.
Claim 3. The method of claim 2, wherein said anti-B7-H3 antibody, or antigen-binding fragment thereof:
(a) competes for B7-H3 binding with BRCA84D, BRCA69D, PRCA157, or with an anti-B7-H3 antibody selected from Table 5; or
(b) has the three heavy chain CDRs and the three light chain CDRs of BRCA84D, hBRCA84D (1.1), hBRCA84D (2.2), hBRCA84D-2, hBRCA69D (1.1), hBRCA (2.2), or the three heavy chain CDRs and the three light chain CDRs of an anti-B7-H3 antibody selected from Table 5; or
(c) has the heavy chain variable domain and the light chain variable domain of BRCA84D, hBRCA84D (1.1), hBRCA84D (2.2), hBRCA84D-2, hBRCA69D (1.1), hBRCA (2.2), or the heavy chain variable domain and the light chain variable domain of an anti-B7-H3 antibody selected from Table 5.
Claim 4. The method of any one of claims 2-3, wherein said anti-CTLA-4 antibody, or antigen-binding fragment thereof:
(a) competes for CTLA-4 binding with ipilimumab, tremelimumab, or with an anti-CTLA-4 antibody selected from Table 6; or
(b) has the three heavy chain CDRs and the three light chain CDRs of nivolumab, ipilimumab, tremelimumab, or the three heavy chain CDRs and the three light chain CDRs of an anti-CTLA-4 antibody selected from Table 6; or (c) has the heavy chain variable domain and the light chain variable domain of nivolumab, ipilimumab, tremelimumab, or the heavy chain variable domain and the light chain variable domain of an anti-CTLA-4 antibody selected from Table 6.
The method of any one of claims 2-4, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises an Fc Domain, and said anti- CTLA-4 antibody or antigen-binding fragment thereof comprises an Fc Domain.
The method of 5, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof, comprises a variant Fc Domain having at least one modification in said Fc Domain that enhances ADCC.
The method of any one of claims 2-6, wherein said anti-B7-H3 antibody is hBRCA84D-2 and said anti-CTLA-4 antibody is ipilimumab, or tremelimumab.
The method of any one of claims 2-8, wherein said method comprises an Induction Period followed by a Maintenance Period, wherein:
(I) during said Induction Period, the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight every three weeks, such that a total of four doses of the anti-CTLA-4 antibody is administered and a total of no more than 10 doses of the anti-B7-H3 antibody is administered, and
(II) during the Maintenance Period, either:
(A) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight, without any administration of the anti- CTLA-4 antibody; or
(B) the anti-B7-H3 antibody is administered at a dosage of 1 to 15 mg/kg body weight weekly, and the anti-CTLA-4 antibody is administered at a dosage of 1 to 15 mg/kg body weight once every twelve weeks, for the duration of such Maintenance Period. Claim 9. The method of claim 8, wherein said anti-B7-H3 antibody is administered at a dosage of 1 mg/kg body weight, 3 mg/kg body weight, 10 mg/kg body weight or 15 mg/kg body weight.
Claim 10. The method of claim 8 or 9, wherein said anti-B7-H3 antibody is administered at a dosage of 1 mg/kg body weight, 3 mg/kg body weight, 10 mg/kg body weight or 15 mg/kg body weight, and said anti-CTLA-4 is administered at a dosage of 1 mg/kg body weight, 3 mg/kg body weight, 6 mg/kg body weight, 10 mg/kg body weight, or 15 mg/kg body weight.
Claim 11. The method of claim 9 or 10, wherein said anti-B7-H3 antibody is administered at a dosage of 3 mg/kg, 10 mg/kg or 15 mg/kg and said anti-CTLA-4 is administered at a dosage of 3 mg/kg.
Claim 12. The method of any one of claims 2-11, wherein the anti-B7-H3 antibody and the anti-CTLA-4 antibody are administered by IV infusion.
Claim 13. The method of any one of claims 2-12, wherein every three or twelve weeks said anti-B7-H3 antibody and said anti-CTLA-4 antibody are administered within a 48-hour period.
Claim 14. The method of any one of claims 1 to 13, wherein said cancer is a B7-H3- expressing cancer.
Claim 15. The method of claim 14, wherein said B7-H3-expressing cancer is a squamous cell cancer of the head and neck (SCCHN), a bladder cancer, a breast cancer, a colorectal cancer, a gastric cancer, a glioblastoma, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, a pharyngeal cancer, a prostate cancer, a renal cell carcinoma, a small round blue cell tumor, a neuroblastoma, or a rhabdomyosarcoma, in which B7-H3 is expressed.
Claim 16. The method of any one of claims 1 to 15, wherein said treatment further comprises the step of administering a third therapeutic agent, wherein said third therapeutic agent is selected from the group consisting of an anti-angiogenic agent, an anti-neoplastic agent, a chemotherapeutic agent, and a cytotoxic agent. Claim 17. The method of any one of claims 2-16, wherein said anti-B7-H3 antibody is hBRCA84D-2.
Claim 18. The method of any one of claims 2-17, wherein said anti-CTLA-4 antibody is ipilimumab.
Claim 19. The method of any one of claims 2-17, wherein said anti-CTLA-4 antibody is tremelimumab.
Claim 20. The method of any one of claims 2-17, wherein said anti-CTLA-4 antibody is an anti-CTLA-4 antibody selected from Table 6.
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