WO2017034906A1 - Anticorps monoclonal anti-hla-dr à administrer par voie sous-cutanée pour le traitement de tumeurs malignes hématologiques - Google Patents

Anticorps monoclonal anti-hla-dr à administrer par voie sous-cutanée pour le traitement de tumeurs malignes hématologiques Download PDF

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WO2017034906A1
WO2017034906A1 PCT/US2016/047483 US2016047483W WO2017034906A1 WO 2017034906 A1 WO2017034906 A1 WO 2017034906A1 US 2016047483 W US2016047483 W US 2016047483W WO 2017034906 A1 WO2017034906 A1 WO 2017034906A1
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antibody
hla
seq
antibodies
cell
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PCT/US2016/047483
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WO2017034906A8 (fr
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David M. Goldenberg
William A. Wegener
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Immunomedics, Inc.
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Priority claimed from US14/876,200 external-priority patent/US9683050B2/en
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Priority to CA2987644A priority Critical patent/CA2987644A1/fr
Priority to EP16839842.8A priority patent/EP3337508A4/fr
Publication of WO2017034906A1 publication Critical patent/WO2017034906A1/fr
Publication of WO2017034906A8 publication Critical patent/WO2017034906A8/fr

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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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • 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
    • 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
    • C07K16/2833—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 MHC-molecules, e.g. HLA-molecules
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention concerns improved methods of treating hematologic cancers by subcutaneous administration of an anti-HLA-DR antibody or antigen-binding fragment thereof.
  • the anti-HLA-DR antibody or fragment thereof is a humanized L243 (hL243) antibody, as disclosed in the Examples below. More preferably, the hL243 antibody is an IgG4 antibody, with decreased ADCC and CDC. Most preferably, the hL243 antibody comprises a Ser241Pro point mutation in the hinge region of the antibody or fragment thereof.
  • the anti-HLA-DR antibody is prepared in a concentrated formulation that allows for subcutaneous administration of small volumes of solution.
  • a dosage of 200 mg of antibody is administered once, twice, or three times a week for the first three weeks of a 4-week cycle.
  • Patients may receive two or more consecutive treatment cycles, followed by maintenance therapy (e.g., one week of treatment every four weeks times 4).
  • maintenance therapy e.g., one week of treatment every four weeks times 4.
  • administration induces no more than a Grade 3 or Grade 4 toxicity.
  • administration results in a decrease in tumor size for solid tumors, or a decrease in white blood cell count for non-solid tumors. Decreases in tumor size preferably are in the range of a 40% to 90% reduction in tumor volume.
  • the antibody or fragment may be administered alone, as a conjugate of a therapeutic agent, or in combination with one or more different therapeutic agents, as discussed in detail below.
  • the therapeutic agent is a Bruton kinase inhibitor (such as ibrutinib) or a PI3K inhibitor (such as idelalisib).
  • a Bruton kinase inhibitor such as ibrutinib
  • a PI3K inhibitor such as idelalisib
  • the combination of antibody and therapeutic agent preferably exhibits synergistic effects in treating hematologic cancers.
  • Rituximab anti-CD20 IgG therapy is credited with revitalizing antibody therapies with its ability to effectively treat follicular lymphoma without the extensive side effects associated with more traditional chemotherapy regimens. Since rituximab' s approval by the FDA in 1997, the mortality rate from NHL has declined by 2.8% per year (Molina, 2008, Ann Rev Med 59:237-50), and the use of this agent has been expanded to a variety of diseases.
  • Rituximab was less effective in the more aggressive types of NHL, such as diffuse large B cell lymphoma (DLBCL), but when it was combined with combination chemotherapy, improved and durable objective responses compared to the separate therapies were found, making R-CHOP a standard protocol for the treatment of DLBCL (e.g., Leonard et al., 2008, Semin Hematol 45:S11-16; Friedberg et al., 2002, Br J Haematol 117:828-34).
  • DLBCL diffuse large B cell lymphoma
  • alemtuzumab for chronic lymphocytic leukemia (CLL)
  • CLL chronic lymphocytic leukemia
  • HLA-DR human leukocyte antigen-DR
  • MHC major histocompatibilty complex
  • the human HLA-DR antigen is expressed in non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), and other B-cell malignancies at significantly higher levels than typical B-cell markers, including CD20.
  • NHL non-Hodgkin lymphoma
  • CLL chronic lymphocytic leukemia
  • CD20 other B-cell malignancies at significantly higher levels than typical B-cell markers, including CD20.
  • Preliminary studies indicate that anti-HLA-DR mAbs are markedly more potent than other naked mAbs of current clinical interest in in vitro and in vivo experiments in lymphomas, leukemias, and multiple myeloma (Stein et al., unpublished results).
  • HLA-DR is also expressed on a subset of normal immune cells, including B cells, monocytes/macrophages, Langerhans cells, dendritic cells, and activated T cells (Dechant et al., 2003, Semin Oncol 30:465-75).
  • B cells normal immune cells
  • monocytes/macrophages including B cells, monocytes/macrophages, Langerhans cells, dendritic cells, and activated T cells.
  • the L243 antibody (hereafter mL243) is a murine IgG2a anti-HLA-DR antibody.
  • This antibody may be of potential use in the treatment of diseases such as autoimmune disease or cancer, particularly leukemias or lymphomas, by targeting the D region of HLA.
  • mL243 demonstrates potent suppression of in vitro immune function and is monomorphic for all HLA-DR proteins.
  • problems exist with the administration of mouse antibodies to human patients, such as the induction of a human anti-mouse antibody (HAMA) response.
  • HAMA human anti-mouse antibody
  • the present invention relates to methods of treating hematologic cancer, autoimmune disease or immune dysfunction disease (e.g., GVHD, organ transplant rejection) by subcutaneous administration of an ant-HLA-DR antibody.
  • hematologic cancer e.g., GVHD, organ transplant rejection
  • immune dysfunction disease e.g., GVHD, organ transplant rejection
  • the antibody is chimeric, humanized or human.
  • the anti-HLA-DR antibody competes for binding to, or binds to the same epitope of HLA-DR as, a murine monoclonal antibody mL243 comprising the murine L243 heavy chain CDR sequences CDRl (NYGMN (SEQ ID NO: 39)), CDR2 (WINTYTREPTYADDFKG (SEQ ID NO: 40)) and CDR3 (DITAVVPTGFDY (SEQ ID NO: 41)) and the light chain CDR sequences CDRl
  • the murine L243 antibody of use for competitive binding studies is publicly available from the American Type Culture Collection, Rockville, MD, (see Accession number ATCC HB55).
  • the anti-HLA-DR antibody comprises the L243 heavy chain CDR sequences CDRl (NYGMN (SEQ ID NO: 39)), CDR2 (WINTYTREPTYADDFKG (SEQ ID NO: 40)) and CDR3 (DITAVVPTGFDY (SEQ ID NO: 41)) and the light chain CDR sequences CDRl (RASENIYSNLA (SEQ ID NO: 42)), CDR2 (AASNLAD (SEQ ID NO: 43)), and CDR3 (QHFWTTPWA (SEQ ID NO: 44)).
  • a humanized anti-HLA-DR antibody may further comprise one or more of framework residues 27, 38, 46, 68 and 91 substituted from the mL243 heavy chain and/or one or more of framework residues 37, 39, 48 and 49 substituted from the mL243 light chain.
  • the hL243 antibody comprises the sequences of SEQ ID NO:36 and SEQ ID NO:38.
  • the anti-HLA-DR antibody may be a naked antibody or an immunoconjugate that is attached to at least one therapeutic agent. Conjugates with multiple therapeutic agents of the same or different type are also encompassed.
  • the anti-HLA-DR antibody may be administered in combination with at least one therapeutic agent administered before, simultaneously with or after the anti-HLA-DR antibody.
  • Any therapeutic agent known in the art, as discussed in more detail below, may be utilized in combination with or attached to the anti-HLA-DR antibody, including but not limited to radionuclides, immunomodulators, anti-angiogenic agents, cytokines, chemokines, growth factors, hormones, drugs, prodrugs, enzymes, oligonucleotides, siRNAs, pro-apoptotic agents, photoactive therapeutic agents, cytotoxic agents, chemotherapeutic agents, toxins, Bruton kinase inhibitors, PI3K inhibitors, and other antibodies or antigen binding fragments thereof.
  • the subject antibody may bind to at least one epitope of HLA-DR on HLA-DR + cells, resulting in cell death.
  • cell death may result without use of either cytotoxic addends or immunological effector mechanisms, for example by induction of apoptosis.
  • the anti-HLA-DR antibodies may be of use for therapy of any disease state in which HLA-DR + cells are involved, including but not limited to various forms of cancer or autoimmune disease.
  • the subject antibodies may be used in a pharmaceutical composition for therapeutic and/or diagnostic use.
  • a pharmaceutical composition may contain further therapeutic agents as described below, in addition to other standard components such as buffers, detergents, salts, excipients, preservatives and other such agents known in the art.
  • the composition may be a high- concentration formulation, as disclosed for example in U.S. Patent Nos. 8,658,773 and 9, 180,205, incorporated herein by reference.
  • the pharmaceutical composition or fusion protein or multispecific antibody may further comprise one or more additional antibodies or fragments thereof which bind to an antigen selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein (AFP), a-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3- antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95
  • CDKN2A CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1 a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gplOO, GRO- ⁇ , HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, la, IGF-1R, IFN- ⁇ , IFN-a, IFN- ⁇ , IFN-.lamda., IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,
  • any pharmaceutical composition containing a humanized anti -HLA-DR antibody is administered before, with, or after any pharmaceutical composition containing a humanized anti -HLA-DR antibody.
  • Exemplary additional antibodies that may be utilized in combination with an anti- HLA-DR include, but are not limited to, hRl (anti-IGF-lR, U.S. Patent Application Serial No. 13/688,812, filed 11/29/12) hPAM4 (anti-mucin, U.S. Patent No. 7,282,567), hA20 (anti- CD20, U.S. Patent No. 7,151, 164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hFMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLLl (anti-CD74, U.S. Patent No.
  • Various embodiments may concern use of the subject anti-HLA-DR antibodies or fragments thereof to treat or diagnose a disease, including but not limited to B cell non- Hodgkin's lymphomas, B cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T cell lymphomas and leukemias, multiple myeloma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, and skin cancers.
  • a disease including but not limited to B cell non- Hodgkin's lymphomas, B cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T cell lymphomas and leukemias, multiple myeloma, Waldenstrom's macro
  • the carcinomas may be selected from the group consisting of carcinomas of the oral cavity, gastrointestinal tract, pulmonary tract, breast, ovary, prostate, uterus, urinary bladder, pancreas, liver, gall bladder, skin, and testes.
  • the subject anti-HLA-DR antibodies or fragments may be used to treat an autoimmune disease, for example acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease
  • the subject antibodies may be used to treat leukemia, such as chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or acute myeloid leukemia.
  • the antibody or fragment thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject.
  • Preferred allotypes for administration include a non-Glml allotype (nGlml), such as Glm3, Glm3,l, Glm3,2 or Glm3, l,2. More preferably, the allotype is selected from the group consisting of the nGlml, Glm3, nGlml,2 and Km3 allotypes.
  • FIG. 1 illustrates an exemplary DNA encoding an amino acid sequence V K of the mouse L243 anti-HLA-DR antibody.
  • the putative CDR regions are underlined. Nucleotide residues are numbered sequentially.
  • Rabat's Ig molecule numbering is used for amino acid residues.
  • the numbering for the residues with a letter (on top) is the number of preceding residues plus the letter, eg, the number for T following N52 is 52A; the numbers for N, N and L following 82 are 82A, 82B and 82C, respectively.
  • FIG. 2 illustrates an exemplary DNA encoding an amino acid sequence V H of the mouse L243 anti-HLA-DR antibody.
  • the putative CDR regions are underlined. Nucleotide residues are numbered sequentially.
  • Rabat's Ig molecule numbering is used for amino acid residues as described above.
  • FIG. 3 illustrates exemplary DNA and amino acid sequences of a humanized L243 V K .
  • the bold and underlined sections of the amino acid sequences indicate the CDRs as defined by the Kabat numbering scheme.
  • FIG. 4 illustrates exemplary DNA and amino acid sequences of a humanized L243 V H .
  • the bold and underlined sections of the amino acid sequences indicate the CDRs as defined by the Kabat numbering scheme.
  • FIG. 5 illustrates an exemplary antigen-binding specificity of hL243.
  • Raji cells preincubated with a saturating concentration of mL234 (for blocking cell surface antigen ("Ag") sites) or without, were resuspended in PBS containing 1% BSA and 10 ⁇ g/ml of purified hL243 and incubated for 1 h at 4°C. After washing, the cells were resuspended in PBS containing 1% BSA and PE-labeled goat anti-human IgG, Fc fragment specific antibody. After further incubation at 4°C for 30 min, the cells were counted in a Guava PCA.
  • the left side of the Figure shows specific binding of hL243 to Raji human lymphoma cells, which was blocked by preincubation of the cells with mL243.
  • the right side of the Figure shows a negative binding control, performed with anti-CEA antibody (hMN-14) in place of hL243 under identical conditions.
  • FIG. 6 illustrates exemplary Ag-binding affinities comparing hL243 ⁇ 4P and mL243 in a competitive cell surface binding assay.
  • a constant amount (100,000 cpm, -10 ⁇ / ⁇ ) of 125 I-labeled mL234 (on left) or hL243y4P (on right) was mixed with varying concentrations (0.2-700 nM) of unlabeled hL243y4P (solid triangle) or mL2343 (solid box).
  • the mixtures were added to Raji cells and incubated at room temperature for 2 h.
  • the cells were washed to remove unbound antibodies and the radioactivity associated with the cells was counted.
  • hL243y4P and mL234 were shown to compete with each other for binding to cell surface Ag. In both cases hL243y4P appeared to bind to Raji cells more strongly than mL243.
  • FIG. 7 illustrates exemplary Ag-binding affinities of hL243y4P and mL243 determinated by direct cell surface saturation binding and Scachard plot analysis. Varying concentrations of 125 I-labeled mL234 (solid square) or hL243y4P (open triangle) were incubated with 2xl0 5 Daudi human lymphoma cells at 4°C for 2 h, and unbound radioactivity was removed from cell suspensions by washing. The cell-associated radioactivity was counted, specific binding of radiolabeled antibody to the cell surface antigen calculated, and Scatchard plot analysis was then applied to determine the maximum number of binding sites per cell and the apparent antigen-binding affinity constant.
  • the maximum binding of mL234 or hL243y4P to Daudi cell surface was 6xl0 6 molecules/cell.
  • the dissociation constants determined for mL234 or hL243y4P were 14 and 2.6 nM, respectively.
  • FIG. 8 illustrates that hL243 is effective in killing target cells in the presence of human serum complement. Daudi cells were incubated with hL243, hL243y4P, hA20 (a positive control), and hMN-14 (a negative control) in the presence of human serum complement. hL243y4P was shown not to produce any complement-induced cytotoxicity.
  • FIG. 9A illustrates LDH release by ADCC as observed for hL243, hL243y4P, hA20 (positive control) and hMN-14 (negative control).
  • FIG. 9B illustrates % cell lysis by ADCC as observed for hL243, hL243y4P, hA20 (positive control) and hMN-14 (negative control).
  • FIG. 10 illustrates exemplary in vitro proliferative assays on Daudi (top) and Raji (bottom) cell lines at the end of 2 days.
  • FIG. 11A illustrates exemplary in vitro proliferative assays on Daudi cell lines at the end of 3 days.
  • FIG. 11B illustrates exemplary in vitro proliferative assays on Raji cell lines at the end of 3 days.
  • FIG. 12 illustrates exemplary median survival times for tumor-bearing SCID mice injected with hL243y4P.
  • FIG. 13 illustrates comparative induction of apoptosis in dog lymphoma cells (measured as % cells with a sub GO/Gl phase DNA content) caused by L243, hL243 (IgG4 isotype), hMN-14 (humanized MN-14 IgG), and Ag8 (murine myeloma derived mAb).
  • L243 and hL243 caused apoptosis when crosslinked with goat anti-mouse (GAM) and goat-anti human (GAH) antibodies respectively.
  • FIG. 14A illustrates anti-proliferative effects of humanized antibodies (hLLl, hLL2, Rituximab, hA2, hMN-14 and hL243 IgG4 isotype), with and without goat anti-human IgG (GAH)) on Namalwa human B cell lymphoma cell line as determined by a 3 H-thymidine uptake assay with single antibodies.
  • humanized antibodies hLLl, hLL2, Rituximab, hA2, hMN-14 and hL243 IgG4 isotype
  • FIG. 14B illustrates anti-proliferative effects of humanized antibodies (hLLl, hLL2, Rituximab, hA2, hMN-14 and hL243 IgG4 isotype), with and without goat anti-human IgG (GAH)) on Namalwa human B cell lymphoma cell line as determined by a 3 H-thymidine uptake assay with mixtures of antibodies.
  • humanized antibodies hLLl, hLL2, Rituximab, hA2, hMN-14 and hL243 IgG4 isotype
  • FIG. 15A illustrates CDC assays in Raji cells when exposed to various antibodies disclosed herein.
  • FIG. 15B illustrates CDC assays in Ramos cells when exposed to various antibodies disclosed herein.
  • FIG. 15C illustrates CDC assays in Namalwa cells when exposed to various antibodies disclosed herein.
  • FIG. 16 illustrates ADCC assays and calcein AM release when SU-DHL-6 cells are exposed to various antibodies disclosed herein.
  • FIG. 17A illustrates anti-proliferative effects of hL243y4P on several cell lines disclosed in MTT studies.
  • FIG. 17B illustrates anti-proliferative effects of hL243y4P on several cell lines disclosed in 3 H-thymidine uptake assays.
  • hL243 refers to the ⁇ 4 ⁇ form of the antibody.
  • FIG. 18A illustrates induction of apoptosis. Dead cells are represented by clear and apoptotic cells are represented by solid bars. The Figure shows measurement of Sub G DNA in SU-DHL-6 and Namalwa cells. Cells used had 97% viability prior to treatment.
  • FIG. 18B illustrates induction of apoptosis. Dead cells are represented by clear and apoptotic cells are represented by solid bars. The Figure shows Annexin V/7-ADD at 4 and 24 hours. Cells used had 97% viability prior to treatment.
  • FIG. 19 illustrates mitochondrial membrane potential using a JC-1 assay in several cell lines.
  • FIG. 20A illustrates cleaved caspase-3 time course studies in Daudi cells.
  • FIG. 20B illustrates P-AKT time course studies in Daudi cells.
  • FIG. 21 illustrates therapy of Raji-bearing SCID mice with murine L243 and
  • FIG. 22 A In vitro effects of murine L243 on canine lymphoma aspirates. L234 binding to the aspirates from 4 dogs. White bars, Ag8; gray bars, L243.
  • FIG. 22B In vitro effects of murine L243 on canine lymphoma aspirates. Percent apoptotic cells as measured by flow cytometry of hypodiploid DNA (sub GO) following propidium iodine staining. Incubations were performed without second antibody or in the presence of goat anti-mouse IgG: white bars, Ag8 without second antibody; striped bars, Ag8 with GAM; gray bars, L243 without second antibody; black bars, L243 with GAM; *, O.05 vs. Ag8.
  • FIG. 22C In vitro effects of murine L243 on canine lymphoma aspirates. Viable cell count was performed on two of the specimens by flow cytometry analysis of the cell count within a viable gate defined in the forward scatter vs. side scatter dot plot: white bars, Ag8 without second antibody; striped bars, Ag8 with GAM; gray bars, L243 without second antibody; black bars, L243 with GAM; *, O.05 vs. Ag8.
  • FIG. 23 A In vitro effects of IMMU-114 on canine lymphoma aspirates. Percent apoptotic cells as measured by flow cytometry of hypodiploid DNA (sub GO) following propidium iodine staining. Incubations were performed without second antibody or in the presence of goat anti-mouse IgG (GAM) or goat anti-human IgG (GAH). Error bars, SD of three replicates. *, significant change ( ⁇ 0.05) relative to no mAb control.
  • FIG. 23B In vitro effects of FMMU-114 on canine lymphoma aspirates. Percent specific lysis in CDC assays on aspirate of dog #171205. Error bars, SD of three replicates. *, significant change ( ⁇ 0.05) relative to no mAb control.
  • FIG. 23C In vitro effects of IMMU-114 on canine lymphoma aspirates. Percent specific lysis in ADCC assays on aspirate of dog #171205. Error bars, SD of three replicates. *, significant change ( ⁇ 0.05) relative to no mAb control. [053] FIG. 24. Peripheral blood lymphocyte count and lymphocyte subset phenotyping indicated a decrease in both B- and T-cell lymphocytes. ⁇ , total lymphocyte count; ⁇ , T-cell count, relative to baseline; O, B cell count, relative to baseline.
  • FIG. 25 Clearance of L243 in a dog with lymphoma (upper) and of IMMU-114 in two normal dogs (middle and lower).
  • the L243 doses administered in the dog with lymphoma (upper) were 1.5 mg/kg for treatment 1 and 3.0 mg/kg for the remaining 3 treatments.
  • EVIMU-l 14 doses were 3.0 mg/kg for the initial dose in both normal dogs (middle and lower).
  • the second dose of IMMU-114 administered to second normal dog (lower) was 1.3 mg/kg.
  • FIG. 26 Effect of different specificity antibodies on survival. 250 ⁇ g of the indicated antibodies was injected twice per week for 4 weeks, starting 1 day after injection of WSU-FSCCL tumor cells.
  • FIG. 27 Ex vivo effects of mAbs on whole blood. Heparinized whole blood of healthy volunteers was incubated with mAbs then assayed by flow cytometry. Data are shown as % of untreated control. Error bars, SD of 3 replicates. *, P ⁇ 0.05 relative to no mAb control.
  • FIG. 28 Effect of ERK, INK and ROS inhibitors on hL234g4P mediated apoptosis in Raji cells.
  • FIG. 29 Tumor reduction in a patient with follicular lymphoma treated with anti- HLA-DR antibody.
  • ADCC antibody-dependent cell mediated cytotoxicity
  • nonspecific cytotoxic cells that express Fc receptors (natural killer cells, neutrophils, and macrophages) recognize bound antibody on target cells and subsequently cause lysis of the target cells.
  • the primary cells for mediating ADCC are the natural killer cells (express the FcDRIII only) and monocytes (express FcDRI, FcDRII and FcDRIII).
  • “Complement-dependent cytotoxicity” or “CDC” refers to the lysing of a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (eg, an antibody) complexed with a cognate antigen.
  • Fc receptor or "FcR” is used to describe a receptor that binds to the Fc region of an antibody.
  • Both CDC and ADCC require the Fc portion of a MAb and the effect of ADCC can be augmented by increasing the binding affinity for FcyR (IgG Fc receptors) on effector cells (Shinkawa, et al, J Biol Chem 278: 3466-3473, 23; Shields et a/, J Biol Chem 211: 26733-2674, 22; Shields et al, J Biol Chem 276: 6591-664, 22; Davies et al, Biotechnol Bioeng 74: 288-294,21; and Umana et al, Nature Biotechnol 176-18, 1999).
  • an “antibody” as used herein refers to a full-length (ie, naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (eg, an IgG antibody) or an immunologically active (ie, specifically binding) portion of an immunoglobulin molecule, like an antibody fragment.
  • An “antibody” includes monoclonal, polyclonal, bispecific, multispecific, murine, chimeric, humanized and human antibodies.
  • a "naked antibody” is an antibody or antigen binding fragment thereof that is not attached to a therapeutic or diagnostic agent.
  • the Fc portion of an intact naked antibody can provide effector functions, such as complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev 50:59-87, 1998).
  • Other mechanisms by which naked antibodies induce cell death may include apoptosis. (Vaswani and Hamilton, Ann Allergy Asthma Immunol 81 : 105- 119, 1998.)
  • an “antibody fragment” is a portion of an intact antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, sFv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.
  • antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
  • Single-chain antibodies often abbreviated as “scFv” consist of a polypeptide chain that comprises both a V H and a V L domain which interact to form an antigen- binding site.
  • the V H and V L domains are usually linked by a peptide of 1 to 25 amino acid residues.
  • Antibody fragments also include diabodies, triabodies and single domain antibodies (dAb).
  • the subject anti-HLA-DR antibody may be a humanized L243 antibody.
  • Such antibodies bind to the same epitope on HLA-DR as the parental murine L243 antibody, but have reduced immunogenicity.
  • mL243 is a monoclonal antibody previously described by Lampson & Levy (J Immunol, 1980, 125:293). The amino acid sequences of the light and heavy chain variable regions of the mL243 antibody are shown in FIG. 1 and FIG. 2. mL243 has been deposited at the American Type Culture Collection, Rockville, MD, under Accession number ATCC HB55.
  • the humanized L243 antibodies may comprise the L243 heavy chain CDR sequences CDRl (NYGMN (SEQ ID NO: 39)), CDR2 (WINTYTREPTYADDFKG (SEQ ID NO: 40)) and CDR3 (DITAVVPTGFDY (SEQ ID NO: 41)) and the light chain CDR sequences CDRl (RASENIYSNLA (SEQ ID NO: 42)), CDR2 (AASNLAD (SEQ ID NO: 43)), and CDR3 (QHFWTTPWA (SEQ ID NO: 44)), attached to human antibody FR and constant region sequences.
  • one or more murine FR amino acid residues are substituted for the corresponding human FR residues, particularly at locations adjacent to or nearby the CDR residues.
  • Exemplary murine V H residues that may be substituted in the humanized design are at positions: F27, K38, K46, A68 and F91.
  • Exemplary murine V L residues that may be substituted in the humanized design are at positions R37, K39, V48, F49, and Gl .
  • FIG. 3 and FIG. 4 A particularly preferred form of hL243 antibody is illustrated in FIG. 3 and FIG. 4, incorporating FR sequences from the human RF-TS3, NEWM and REI antibodies.
  • the FR residues may be derived from any suitable human
  • the humanized antibody can fold such that it retains the ability to specifically bind HLA-DR.
  • the type of human framework (FR) used is of the same/similar class/type as the donor antibody. More preferably, the human FR sequences are selected to have a high degree of sequence homology with the corresponding murine FR sequences, particularly at positions spatially close or adjacent to the CDRs.
  • the frameworks (ie, FR1-4) of the humanized L243 V H or V L may be derived from a combination of human antibodies. Examples of human
  • CDR-grafted humanized antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI, RF and EU.
  • human RF-TS3 FR1-3 and NEWM FR4 are used for the heavy chain and REI FR1-4 are used for the light chain.
  • the variable domain residue numbering system used herein is described in Kabat et al, (1991), Sequences of Proteins of Immunological Interest, 5th Edition, United States Department of Health and Human Services
  • the light and heavy chain variable domains of the humanized antibody molecule may be fused to human light or heavy chain constant domains.
  • the human constant domains may be selected with regard to the proposed function of the antibody. In one embodiment, the human constant domains may be selected based on a lack of effector functions.
  • the heavy chain constant domains fused to the heavy chain variable region may be those of human IgA (od or a2 chain), IgG ( ⁇ , ⁇ 2, ⁇ 3 or ⁇ 4 chain) or IgM ( ⁇ chain).
  • the light chain constant domains which may be fused to the light chain variable region include human lambda and kappa chains.
  • a ⁇ chain is used.
  • a ⁇ 4 chain is used.
  • the use of the ⁇ 4 chain may in some cases increase the tolerance to hL243 in subjects (decreased side effects and infusion reactions, etc).
  • analogues of human constant domains may be used. These include but are not limited to those constant domains containing one or more additional amino acids than the corresponding human domain or those constant domains wherein one or more existing amino acids of the corresponding human domain have been deleted or altered. Such domains may be obtained, for example, by oligonucleotide directed mutagenesis.
  • an anti-HLA-DR antibody or fragment thereof may be a fusion protein.
  • the fusion protein may contain one or more anti-HLA-DR antibodies or fragments thereof.
  • the fusion protein may also comprise one or more additional antibodies against a different antigen, or may comprise a different effector protein or peptide, such as a cytokine.
  • the different antigen may be a tumor marker selected from a B cell lineage antigen, (eg, CD 19, CD20, or CD22) for the treatment of B cell malignancies.
  • the different antigen may be expressed on other cells that cause other types of malignancies.
  • the cell marker may be a non-B cell lineage antigen, such as selected from the group consisting of HLA-DR, CD3, CD33, CD52, CD66, MUC1 and TAC.
  • an anti-HLA-DR antibody may be combined with other antibodies and used to treat a subject having or suspected of developing a disease.
  • an anti-HLA-DR antibody or fragment thereof may be combined with an anticancer monoclonal antibody such as a humanized monoclonal antibody ⁇ eg hA20, anti-CD20 Mab) and used to treat cancer.
  • an anti- HLA-DR antibody may be used as a separate antibody composition in combination with one or more other separate antibody compositions, or used as a bi-functional antibody containing, for example, one anti-HLA-DR and one other anti-tumor antibody, such as hA20.
  • the antibody may target a B cell malignancy.
  • the B cell malignancy may consist of indolent forms of B cell lymphomas, aggressive forms of B cell lymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, Waldenstrom's macroglobulinemia, and multiple myeloma.
  • Other non-malignant B cell disorders and related diseases that may be treated with the subject antibodies include many autoimmune and immune dysregulatory diseases, such as septicemia and septic shock.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • the antigen is a human antigen.
  • MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al, "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR
  • BIOLOGY, VOL. 10, pages 79-104 The Humana Press, Inc. 1992.
  • the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. The use of antibody components derived from humanized, chimeric or human antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • a chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody.
  • Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject.
  • CDRs complementarity-determining regions
  • a chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the
  • variable domains of a human antibody The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences.
  • additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest et al., Biotechnology 9:266 (1991) and Verhoeyen et al, Science 239: 1534 (1988).
  • those human FR amino acid residues that differ from their murine counterparts and are located close to or touching one or more CDR amino acid residues would be candidates for substitution.
  • the claimed methods and procedures may utilize human antibodies produced by such techniques.
  • the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40).
  • Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
  • the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
  • Fab fragment antigen binding protein
  • RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).
  • Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22).
  • Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library.
  • libraries may be screened by standard phage display methods, as known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43 : 159- 162).
  • Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3 :5564-571 (1993). Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos.
  • transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols.
  • Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 3(55:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
  • a non- limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 11-23) from Abgenix (Fremont, CA).
  • the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
  • the XenoMouse® was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
  • the human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques.
  • a XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
  • a variety of strains of XenoMouse® are available, each of which is capable of producing a different class of antibody.
  • Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
  • the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
  • Antibody fragments which recognize specific epitopes can be generated by known techniques.
  • Antibody fragments are antigen binding portions of an antibody, such as F(ab') 2, Fab', F(ab) 2 , Fab, Fv, sFv and the like.
  • F(ab') 2 fragments can be produced by pepsin digestion of the antibody molecule and Fab ' fragments can be generated by reducing disulfide bridges of the F(ab') 2 fragments.
  • Fab ' expression libraries can be constructed (Huse et al., 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.
  • F(ab) 2 fragments may be generated by papain digestion of an antibody and Fab fragments obtained by disulfide reduction.
  • a single chain Fv molecule comprises a VL domain and a VH domain.
  • the VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L).
  • L peptide linker
  • DABs single domain antibodies
  • An antibody fragment can be prepared by proteolytic hydrolysis of the full length antibody or by expression in E. coli or another host of the DNA coding for the fragment.
  • An antibody fragment can be obtained by pepsin or papain digestion of full length antibodies by conventional methods. These methods are described, for example, by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et al, Arch Biochem. Biophys.
  • Antibodies of use may be commercially obtained from a wide variety of known sources.
  • a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • ATCC American Type Culture Collection
  • VA Manassas
  • a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos.
  • antibody sequences or antibody- secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
  • the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
  • Exemplary antibodies that may be utilized include, but are not limited to, hRl (anti- IGF-1R, U.S. patent application Ser. No. 13/688,812, filed Nov. 29, 2012), hPAM4 (anti- mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7, 151,164), hA19 (anti- CD19, U.S. Pat. No. 7,109,304), MMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLLl (anti- CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.
  • hMu-9 anti- CSAp, U.S. Pat. No. 7,387,772
  • hL243 anti-HLA-DR, U.S. Pat. No. 7,612,180
  • hMN-14 anti-CEACAM5, U.S. Pat. No. 6,676,924
  • hMN-15 anti-CEACAM6, U.S. Pat. No.
  • hRS7 anti-EGP-1, U.S. Pat. No. 7,238,785
  • hMN-3 anti-CEACAM6, U.S. Pat. No. 7,541,440
  • Abl24 and Abl25 anti-CXCR4, U.S. Pat. No. 7,138,496
  • the antibody is IMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLLl (anti-CD74), hLL2 (anti- CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20 (anti-CD20).
  • the terms epratuzumab and hLL2 are interchangeable, as are the terms veltuzumab and hA20, hL243g4P, hL243gamma4P and IMMU-114.
  • Alternative antibodies of use include, but are not limited to, abciximab (anti- glycoprotein Ilb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti- EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti- CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA), abc
  • antibodies such as CDP571 (Ofei et al., 2011, Diabetes 45:881-85), MT FAI, M2TNFAI, M3TNFAI, M3T FABI, M302B, M303 (Thermo Scientific, Rockford, 111.), infliximab (Centocor, Malvern, Pa.), certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park, 111.), or Benlysta (Human Genome Sciences).
  • CD Cluster Designation
  • the CD66 antigens consist of five different glycoproteins with similar structures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) gene family members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66 antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normal epithelial cells of the digestive tract and tumor cells of various tissues. Also included as suitable targets for cancers are cancer testis antigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007; 120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al., Cancer Genet.
  • CEACAM6 carcinoembryonic antigen
  • Macrophage migration inhibitory factor is an important regulator of innate and adaptive immunity and apoptosis. It has been reported that CD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med 197: 1467-76).
  • the therapeutic effect of antagonistic anti-CD74 antibodies on MIF-mediated intracellular pathways may be of use for treatment of a broad range of disease states, such as cancers of the bladder, prostate, breast, lung, colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma 52: 1446-54); autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (Morand & Leech, 2005, Front Biosci 10: 12-22; Shachar & Haran, 2011, Leuk Lymphoma 52: 1446-54); kidney diseases such as renal allograft rejection (Lan, 2008, Nephron Exp Nephrol.
  • a broad range of disease states such as cancers of the bladder, prostate, breast, lung, colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al., 2004, BMC Cancer 12
  • Anti-TNF-. alpha antibodies are known in the art and may be of use to treat immune diseases, such as autoimmune disease, immune dysfunction (e.g., graft-versus-host disease, organ transplant rejection) or diabetes.
  • autoimmune disease e.g., graft-versus-host disease, organ transplant rejection
  • Known antibodies against TNF-. alpha include the human antibody CDP571 (Ofei et al., 2011, Diabetes 45:881-85); murine antibodies
  • MTNFAI M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, 111.); infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels, Belgium); and adalimumab (Abbott, Abbott Park, 111.).
  • infliximab Certocor, Malvern, Pa.
  • certolizumab pegol UB, Brussels, Belgium
  • adalimumab Abbott, Abbott Park, 111.
  • anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-CD 11a); muromonab-CD3 (anti-CD3 receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-.alpha.4 integrin) and omalizumab (anti-IgE).
  • anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-CD 11a); muromonab-CD3 (anti-CD3 receptor); anti-CD40L (
  • checkpoint inhibitor In contrast to the majority of anti-cancer agents, checkpoint inhibitor do not target tumor cells directly, but rather target lymphocyte receptors or their ligands in order to enhance the endogenous antitumor activity of the immune system. (Pardoll, 2012, Nature Reviews 12:252-264) Because such antibodies act primarily by regulating the immune response to diseased cells, tissues or pathogens, they may be used in combination with other therapeutic modalities, such as the subject anti-HLA-DR antibodies, to enhance their antitumor effect.
  • PD-1 Programmed cell death protein 1
  • CD279 encodes a cell surface membrane protein of the immunoglobulin superfamily, which is expressed in B cells and NK cells (Shinohara et al., 1995, Genomics 23 :704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197: 177-87; Pardoll, 2012, Nature Reviews 12:252-264).
  • Anti-PDl antibodies have been used for treatment of melanoma, non-small-cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple- negative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49: 1089-96;
  • anti-PDl antibodies include lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011, CURETECH LTD.).
  • Anti-PDl antibodies are commercially available, for example from ABCAM® (AB 137132), BIOLEGEND® (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, Jl 16, MIH4).
  • PD-L1 Programmed cell death 1 ligand 1
  • CD274 is a ligand for PD- 1, found on activated T cells, B cells, myeloid cells and macrophages.
  • the complex of PD-1 and PD-L1 inhibits proliferation of CD8+ T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).
  • Anti-PDLl antibodies have been used for treatment of non-small cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematologic malignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res
  • anti-PDLl antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDFMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).
  • Anti-PDLl antibodies are also commercially available, for example from
  • CTLA-4 Cytotoxic T-lymphocyte antigen 4
  • CD 152 Cytotoxic T-lymphocyte antigen 4
  • CTLA-4 acts to inhibit T cell activation and is reported to inhibit helper T cell activity and enhance regulatory T cell immunosuppressive activity (Pardoll, 2012, Nature Reviews 12:252-264).
  • Anti- CTL4A antibodies have been used in clinical trials for treatment of melanoma, prostate cancer, small cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med 11 :89).
  • anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER).
  • Anti-PDl antibodies are commercially available, for example from ABCAM® (AB 134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA1- 35914).
  • Ipilimumab has recently received FDA approval for treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11 :89).
  • checkpoint inhibitor antibodies may be used in combination with anti-HLA-DR antibodies alone or in further combination with an interferon, such as interferon-a, for improved cancer therapy.
  • human HLA-DR antigen a known and well characterized target antigen, such as human HLA-DR.
  • human HLA-DR antigen has been well characterized in the art, for example by its amino acid sequence (see, e.g., GenBank Accession No. ADM15723.1).
  • Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08).
  • the extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21).
  • Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody.
  • the allotypes of IgG antibodies containing a heavy chain ⁇ -type constant region are designated as Gm allotypes (1976, J Immunol 117: 1056-59).
  • Glml For the common IgGl human antibodies, the most prevalent allotype is Glml (Stickler et al., 2011, Genes and Immunity 12:213-21). However, the Glm3 allotype also occurs frequently in Caucasians ⁇ Id.). It has been reported that Glml antibodies contain allotypic sequences that tend to induce an immune response when administered to non-Glml (nGlml) recipients, such as Glm3 patients ⁇ Id). Non-Glml allotype antibodies are not as immunogenic when administered to Glml patients ⁇ Id).
  • the human Glml allotype comprises the amino acids D12 (Kabat position 356) and L14 (Kabat position 358) in the CH3 sequence of the heavy chain IgGl .
  • the nGlml allotype comprises the amino acids E12 and M14 at the same locations. Both Glml and nGlml allotypes comprise an E13 residue in between the two variable sites and the allotypes are sometimes referred to as DEL and EEM allotypes.
  • a non-limiting example of the heavy chain constant region sequences for Glml and nGlml allotype antibodies is shown for the exemplary antibodies rituximab (SEQ ID NO:45) and veltuzumab (SEQ ID NO:46).
  • Veltuzumab heavy chain variable region (SEQ ID ⁇ 6)
  • veltuzumab and rituximab are, respectively, humanized and chimeric IgGl antibodies against CD20, of use for therapy of a wide variety of hematological malignancies and/or autoimmune diseases.
  • Table 1 compares the allotype sequences of rituximab vs. veltuzumab.
  • rituximab (Glml7,l) is a DEL allotype IgGl, with an additional sequence variation at Kabat position 214 (heavy chain CHI) of lysine in rituximab vs. arginine in veltuzumab.
  • veltuzumab is less immunogenic in subjects than rituximab ⁇ see, e.g., Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al., 2009, Blood 113 : 1062-70; Robak & Robak, 2011, BioDrugs 25 : 13-25), an effect that has been attributed to the difference between humanized and chimeric antibodies.
  • the difference in allotypes between the EEM and DEL allotypes likely also accounts for the lower immunogenicity of veltuzumab.
  • the anti-HLA-DR antibodies disclosed herein may be used in combination with another molecule attached to the antibody. Attachment may be either covalent or non-covalent.
  • an anti-HLA-DR antibody may be used in a bi-specific antibody, i.e., an antibody that has two different binding sites, one for HLA-DR antibody and another for a different target antigen, such as a hapten or a disease-associated antigen.
  • a bi-specific antibody i.e., an antibody that has two different binding sites, one for HLA-DR antibody and another for a different target antigen, such as a hapten or a disease-associated antigen.
  • exemplary types of tumors that may be targeted include acute lymphoblastic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid, non- Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer Preferred are tumors that have constitutive expression of HLA-DR.
  • One strategy for use of bi-specific antibodies includes pretargeting methodologies, in which therapeutic agent attached to a targetable construct is administered to a subject after a bi-specific antibody has been administered.
  • Pretargeting methods have been developed to increase the target: background ratios of detection or therapeutic agents Examples of pretargeting and biotin/avidin approaches are described, for example, in Goodwin et al, US Pat No 4,863,713; Goodwin et al, J Nucl Med 29:226, 1988; Hnatowich et al, J Nucl Med 28: 1294, 1987; Oehr et al, J Nucl Med 29:728, 1988; Klibanov et al, J Nucl Med 29: 1951, 1988; Sinitsyn et al, J Nucl Med 3 :66, 1989; Kalofonos et al, J Nucl Med 31 : 1791, 199; Schechter et al, Int J Cancer 48: 167, 1991; Paganelli et al, Cancer Res 51
  • bispecific antibodies and targetable constructs may be of use in treating and/or imaging normal or diseased tissue and organs, for example using the methods described in US Pat Nos 6, 126,916; 5,772,981; 5,746,996; 5,328,679; and
  • the anti-HLA-DR antibody or fragment may be conjugated to one or more therapeutic or diagnostic agents.
  • the therapeutic agents do not need to be the same but can be different, e.g. a drug and a radioisotope.
  • 131 I can be incorporated into a tyrosine of an antibody or fusion protein and a drug attached to an epsilon amino group of a lysine residue.
  • Therapeutic and diagnostic agents also can be attached, for example to reduced SH groups and/or to carbohydrate side chains. Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized.
  • a therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
  • such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancer 56: 244 (1994).
  • SPDP N-succinyl 3-(2-pyridyldithio)propionate
  • the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
  • the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
  • the Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a
  • carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al, J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Patent No. 5,443,953 (1995), Leung et al, U.S. patent No. 6,254,868, incorporated herein by reference in their entirety.
  • the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
  • a chelating agent may be attached to an antibody, antibody fragment or fusion protein and used to chelate a therapeutic or diagnostic agent, such as a radionuclide.
  • exemplary chelators include but are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.
  • Methods of conjugation and use of chelating agents to attach metals or other ligands to proteins are well known in the art (see, e.g., U.S. Patent Application Serial No. 12/112,289, incorporated herein by reference in its entirety).
  • radioactive metals or paramagnetic ions may be attached to proteins or peptides by reaction with a reagent having a long tail, to which may be attached a multiplicity of chelating groups for binding ions.
  • a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chains having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • porphyrins polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for
  • Chelates may be directly linked to antibodies or peptides, for example as disclosed in U.S. Patent 4,824,659, incorporated herein in its entirety by reference.
  • Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such
  • Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223 Ra for RAIT are encompassed.
  • therapeutic agents such as cytotoxic agents, anti- angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, Bruton kinase inhibitors, PI3K inhibitors or other agents may be used, either conjugated to the subject anti-HLA-DR antibodies or separately administered before, simultaneously with, or after the antibody.
  • Drugs of use may possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents and combinations thereof.
  • Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, biyostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide
  • epipodophyllotoxin estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-0-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine,
  • mitoxantrone mithramycin, mitomycin, mitotane, navelbine, nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
  • Toxins of use may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • RNase ribonuclease
  • Chemokines of use may include RANTES, MCAF, MlPl-alpha, MIPl-Beta and IP-10.
  • anti-angiogenic agents such as angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin- 12, IP-10, Gro-B, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K pro
  • Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, an hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are
  • lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
  • interferons-a, - ⁇ or - ⁇ interferons-a, - ⁇ or - ⁇ , and stem cell growth factor, such as that designated "SI factor”.
  • cytokines include growth hormones such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;
  • thyroxine insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • LH luteinizing hormone
  • integrin thrombopoietin
  • nerve growth factors such as NGF-B; platelet-growth factor; transforming growth factors (TGFs) such as TGF- a and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, - ⁇ , and - ⁇ ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin,
  • Radionuclides of use include, but are not limited to- lu In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 67 Cu, 90 Y, 125 I, 131 1, 32 P, 33 P, 47 Sc, l u Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 227 Th, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 21 l Fb.
  • the therapeutic radionuclide preferably has a decay-energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.
  • Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20- 5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
  • beta-particle-emitting nuclides are preferably ⁇ 1,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles.
  • Such radionuclides include, but are not limited to: Dy-152, At-21 1, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decay energies of useful alpha- particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000- 8,000 keV, and most preferably 4,000-7,000 keV.
  • radioisotopes of use include U C, 13 N, 15 0, 75 Br, 198 Au, 224 Ac, 126 I, 133 I, 77 Br, 113m In, 95 Ru, 97 Ru, 103 Ru, 105 Ru, 107 Hg, 203 Hg, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 166 Ho, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 T1, 225 Ac, 76 Br, 169 Yb,
  • Some useful diagnostic nuclides may include F, Fe, Cu, Cu, Cu, Ga, 68 Ga, 86 Y, 89 Zr, 94 Tc, 94m Tc, 99m Tc, or i n In.
  • anti-HLA-DR antibodies such as hL243, may be of use in combination with therapeutic radionuclides for sensitization of tumors to radiation therapy (see, e.g., Allen et al., 2007, Cancer Res.
  • Therapeutic agents may include a photoactive agent or dye.
  • compositions such as fluorochrome, and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy. See Jori et al. (eds ), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain (1986), 22:430. Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. See Mew et al., J. Immunol. (1983), 130: 1473; idem., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83 :8744; idem.,
  • Other useful therapeutic agents may comprise oligonucleotides, especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products, such as bcl-2 or p53.
  • a preferred form of therapeutic oligonucleotide is siRNA.
  • Diagnostic agents are preferably selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a
  • diagnostic agents are well known and any such known diagnostic agent may be used.
  • diagnostic agents may include a radionuclide such as 110 In, U1 ln, 177 Lu,
  • Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).
  • Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III).
  • Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes.
  • Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
  • fluorescent labels are known in the art, including but not limited to fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
  • Chemiluminescent labels of use may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt or an oxalate ester.
  • Various embodiments concern methods of treating a cancer in a subject, such as a mammal, including humans, domestic or companion pets, such as dogs and cats, comprising administering to the subject a therapeutically effective amount of an anti-HLA-DR antibody.
  • the anti-HLA-DR antibody is a humanized L243 antibody, as described in further detail in the Examples below.
  • the anti-HLA-DR antibody is administered subcutaneously as a high concentration formulation of between about 100 mg/ml to 225 mg/ml. Use of high concentration formulations allows subcutaneous administration of low volumes of antibody, preferably between 1 to 3 ml or less.
  • the anti-HLA-DR antibody may be used to treat a
  • hematologic cancer such as indolent forms of B cell lymphomas, aggressive forms of B cell lymphomas, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, Burkitt lymphoma, Waldenstrom's macroglobulinemia, and multiple myeloma.
  • immunological diseases which may be treated with the subject antibodies may include, for example, joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; neurological disease such as multiple sclerosis and myasthenia gravis; pancreatic disease such as diabetes, especially juvenile onset diabetes; gastrointestinal tract disease such as chronic active hepatitis, celiac disease, ulcerative colitis, Crohn's disease, pernicious anemia; skin diseases such as psoriasis or scleroderma; allergic diseases such as asthma and in transplantation related conditions such as graft versus host disease and allograft rejection.
  • joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis
  • neurological disease such as multiple sclerosis and myasthenia gravis
  • pancreatic disease such as diabetes, especially juvenile onset diabetes
  • gastrointestinal tract disease such as chronic active hepatitis, celia
  • anti-HLA-DR antibody can be supplemented by administering concurrently or sequentially a therapeutically effective amount of another antibody that binds to or is reactive with another antigen on the surface of the target cell.
  • Preferred additional MAbs comprise at least one humanized, chimeric or human MAb selected from the group consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUCl, MUC2, MUC3, MUC4, MUC5, la, MIF, HM1.2
  • anti-CD 19, anti-CD20, and anti-CD22 antibodies are known to those of skill in the art. See, for example, Ghetie et al, Cancer Res. 48:2610 (1988); Hekman et al, Cancer Immunol.
  • the anti-HLA-DR antibody therapy can be further supplemented with the
  • CVB is a regimen used to treat non-Hodgkin's lymphoma. Patti et al, Eur. J. Haematol. 51: 18 (1993).
  • Other suitable combination chemotherapeutic regimens are well- known to those of skill in the art. See, for example, Freedman et al, "Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.
  • first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma include C- MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP
  • a useful second generation chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin,
  • cyclophosphamide vincristine, dexamethasone and leucovorin
  • a suitable third generation regimen is MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin).
  • Additional useful drugs include phenyl butyrate, bendamustine, and biyostatin-1.
  • the anti-HLA-DR antibody can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the anti-HLA-DR antibody is combined in a mixture with a pharmaceutically suitable excipient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
  • Other suitable excipients are well-known to those in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
  • the anti-HLA-DR antibody can be formulated for intravenous administration via, for example, bolus injection or continuous infusion.
  • anti-HLA-DR antibody is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
  • the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Control release preparations can be prepared through the use of polymers to complex or adsorb the anti-HLA-DR antibody.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release from such a matrix depends upon the molecular weight of the anti-HLA-DR antibody, the amount of anti-HLA-DR antibody within the matrix, and the size of dispersed particles. Saltzman et al, Biophys. J. 55: 163 (1989); Sherwood et al, supra. Other solid dosage forms are described in Ansel et al, PHARMACEUTICAL
  • the anti-HLA-DR antibody may also be administered to a mammal subcutaneously or even by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. Preferably, the anti-HLA-DR antibody is administered subcutaneously with relatively rapid injection of a small volume of antibody formulation (see, e.g., U.S. Patent No. 8,658,773, incorporated herein by reference).
  • the dosage of an administered anti-HLA-DR antibody for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of anti-HLA-DR antibody that is in the range of from about 1 mg/kg to 25 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate. More preferably, the dosage will be 4 to 18 mg/kg, more preferably 6 to 16 mg/kg, more preferably 8 to 12 mg/kg. A dosage of 1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m 2 for a 1.7-m patient.
  • the dose administered to the subject is 200 mg.
  • the dosage may be repeated as needed, for example, once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as every other week for several months, or monthly or quarterly for many months, as needed in a maintenance therapy.
  • an anti-HLA-DR antibody may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.
  • the construct may be administered twice per week for 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m 2 (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once, twice or even thrice weekly for 3 or more weeks.
  • the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. This is particularly true for maintenance therapy, where lower dosages (e.g. 1, 2, 3, or 4 mg/kg or even lower) may be administered less frequently, for a prolonged period.
  • the anti-HLA-DR antibodys are of use for therapy of cancer.
  • cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies.
  • squamous cell cancer e.g., epithelial squamous cell cancer
  • Ewing sarcoma e.g., Ewing sarcoma
  • Wilms tumor astrocytomas
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head-and-neck cancer.
  • cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
  • Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express HLA-DR.
  • cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
  • Astrocytoma Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
  • Metastatic Occult Primary Squamous Neck Cancer Metastatic Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplasia Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer
  • Pheochromocytoma Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive
  • Neuroectodermal and Pineal Tumors T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • compositions described and claimed herein may be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
  • Dysplastic disorders which can be treated include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epi
  • pseudoachondroplastic spondyloepiphysial dysplasia retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
  • Additional pre-neoplastic disorders which can be treated include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
  • benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia
  • leukoplakia keratoses
  • Bowen's disease keratoses
  • Farmer's Skin Farmer's Skin
  • solar cheilitis solar keratosis
  • the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, lipos
  • lymphangioendotheliosarcoma synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
  • Still other embodiments may concern DNA sequences comprising a nucleic acid encoding an anti-HLA-DR antibody or fusion protein.
  • Fusion proteins may comprise an antibody or fragment attached to a different antibody or fragment or to a therapeutic protein or peptide, such as a cytokine.
  • Various embodiments relate to expression vectors comprising the coding DNA sequences.
  • the vectors may contain sequences encoding the light and heavy chain constant regions and the hinge region of a human immunoglobulin to which may be attached chimeric, humanized or human variable region sequences.
  • the vectors may additionally contain promoters that express the encoded protein(s) in a selected host cell, enhancers and signal or leader sequences. Vectors that are particularly useful are pdHL2 or GS.
  • the light and heavy chain constant regions and hinge region may be from a human EU myeloma immunoglobulin, where optionally at least one of the amino acid in the allotype positions is changed to that found in a different IgGl allotype, and wherein optionally amino acid 253 of the heavy chain of EU based on the EU number system may be replaced with alanine.
  • an IgGl sequence may be converted to an IgG4 sequence.
  • kits containing components suitable for treating or diagnosing diseased tissue in a patient.
  • Exemplary kits may contain at least one or more anti- HLA-DR antibody as described herein.
  • a device capable of delivering the kit components through some other route may be included.
  • One type of device, for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject. Inhalation devices may also be used.
  • a therapeutic agent may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized
  • the kit components may be packaged together or separated into two or more containers.
  • the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
  • a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
  • Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.
  • Another component that can be included is instructions to a person using a kit for its use.
  • the anti-HLA-DR antibodies or fragments may be any one of the anti-HLA-DR antibodies or fragments.
  • DOCK- AND-LOCK® DOCK- AND-LOCK®
  • DNL® DOCK- AND-LOCK®
  • PKA regulatory protein kinase
  • AD anchoring domain
  • AKAPs A-kinase anchoring proteins
  • PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al, J. Biol. Chem. 1968;243 :3763).
  • the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has a and ⁇ isoforms (Scott, Pharmacol. Ther.
  • R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999;6:222). Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al, J. Biol. Chem.
  • AKAP microtubule-associated protein-2
  • the dimerization domain and AKAP binding domain of human Rlla are both located within the same N-terminal 44 amino acid sequence (Newlon et al., Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001;20: 1651), which is termed the DDD herein.
  • Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
  • the dimeric motif of DDD contained in a 2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a 2 and b to form a binary, trimeric complex composed of a 2 b.
  • This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chimura et al, Proc. Natl. Acad. Sci. USA.
  • the anti-HLA-DR MAb DNL constructs may be based on a variation of the a 2 b structure, in which an IgG immunoglobulin molecule (e.g., hL243) is attached at its C-terminal end to two copies of an AD moiety.
  • an IgG immunoglobulin molecule e.g., hL243
  • the AD moiety is attached to the C-terminal end of each light chain.
  • Each AD moiety is capable of binding to two DDD moieties in the form of a dimer.
  • the effector moiety is a protein or peptide, more preferably an antibody, antibody fragment or cytokine, which can be linked to a DDD or AD unit to form a fusion protein or peptide.
  • fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
  • the AD and/or DDD moiety may be attached to either the N-terminal or C-terminal end of an effector protein or peptide.
  • site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity. Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
  • the AD and DDD sequences incorporated into the anti-HLA- DR MAb DNL complex comprise the amino acid sequences of DDD1 (SEQ ID NO: 1) and ADl (SEQ ID NO:3) below.
  • the AD and DDD sequences comprise the amino acid sequences of DDD2 (SEQ ID NO:2) and AD2 (SEQ ID NO:4), which are designed to promote disulfide bond formation between the DDD and AD moieties.
  • SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO: l
  • sequence variants AD and/or DDD moieties may be utilized in construction of the anti-HLA-DR MAb DNL complexes.
  • the structure- function relationships of the AD and DDD domains have been the subject of investigation. (See, e.g., Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276: 17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the entire text of each of which is incorporated herein by reference.)
  • DDD sequence of use for construction of DNL complexes is shown in SEQ ID NO:5, wherein "X" represents a conservative amino acid substitution.
  • Conservative amino acid substitutions are discussed in more detail below, but could involve for example substitution of an aspartate residue for a glutamate residue, or a leucine or valine residue for an isoleucine residue, etc. Such conservative amino acid substitutions are well known in the art.
  • SHIOIPPGLTELLOGYTVEVLROOPPDLVEFAVEYFTRLREARA SEQ ID NO: l
  • AKAP-IS RII selective AD sequence
  • the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD are underlined in SEQ ID NO:3. Therefore, the skilled artisan will realize that variants which may function for D L constructs are indicated by SEQ ID NO:6, where "X" is a conservative amino acid substitution.
  • the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to prepare anti-HLA-DR MAb DNL constructs.
  • Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown in SEQ ID NO:8-10. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AKAP-IS sequence shown in SEQ ID NO:3, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine, as shown in SEQ ID NO:4.
  • Carr et al. examined the degree of sequence homology between different AKAP -binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA Rlla DDD sequence of SEQ ID NO: 1. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP proteins. Thus, a potential DDD sequence is indicated in SEQ ID NO: 17, wherein "X" represents a conservative amino acid substitution.
  • amino acid residues that are highly conserved in the DDD and AD sequences from different proteins are ones that it may be preferred to remain constant in making amino acid substitutions, while residues that are less highly conserved may be more easily varied to produce sequence variants of the AD and/or DDD sequences described herein.
  • sequence variants of the DDD and/or AD moieties in certain embodiments it may be preferred to introduce sequence variations in the antibody moiety or the linker peptide sequence joining the antibody with the AD sequence.
  • sequence variations in the antibody moiety or the linker peptide sequence joining the antibody with the AD sequence in one illustrative example, three possible variants of fusion protein sequences, are shown in SEQ ID NO: 18- 20.
  • the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
  • methods for making monoclonal antibodies against virtually any target antigen are well known in the art. Typically, these result in production of murine antibodies against a target antigen.
  • the antigen-binding specificity of murine monoclonal antibodies is determined largely by the hypervariable complementarity determining region (CDR) sequences.
  • Murine antibodies generally comprise 6 CDR sequences, 3 on the antibody light chain and 3 on the heavy chain.
  • chimeric, humanized or human versions of murine antibodies may be constructed by techniques such as CDR grafting, where the murine CDR sequences are inserted into, for example, human antibody framework and constant region sequences, or by attaching the entire murine variable region sequences to human antibody constant region sequences.
  • the variable region sequences of an antibody may be constructed, for example, by chemical synthesis and assembly of oligonucleotides encoding the entire light and heavy chain variable regions of an antibody.
  • the structural, physical and/or therapeutic characteristics of native, chimeric, humanized or human antibodies, or AD or DDD sequences may be optimized by replacing one or more amino acid residues.
  • the functional characteristics of humanized antibodies may be improved by substituting a limited number of human framework region (FR) amino acids with the corresponding FR amino acids of the parent murine antibody. This is particularly true when the framework region amino acid residues are in close proximity to the CDR residues.
  • the therapeutic properties of an antibody such as binding affinity for the target antigen, the dissociation- or off-rate of the antibody from its target antigen, or even the effectiveness of induction of CDC (complement-dependent cytotoxicity) or ADCC (antibody dependent cellular cytotoxicity) by the antibody, may be optimized by a limited number of amino acid substitutions.
  • the DDD and/or AD sequences used to make the anti- ULA-DR DNL constructs may be further optimized, for example to increase the DDD-AD binding affinity. Potential sequence variations in DDD or AD sequences are discussed above.
  • amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions).
  • conservative amino acid substitutions The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
  • the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157: 105-132).
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the use of amino acids whose hydropathic indices are within ⁇ 2 is preferred, within ⁇ 1 are more preferred, and within ⁇ 0.5 are even more preferred.
  • Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554, 101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.
  • amino acid side chain For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
  • a compact side chain such as glycine or serine
  • an amino acid with a bulky side chain e.g., tryptophan or tyrosine.
  • tryptophan or tyrosine The effect of various amino acid residues on protein secondary structure is also a
  • arginine and lysine glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp.
  • conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and He; He and Val; Phe and Tyr.
  • amino acid substitutions In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.
  • ionic bonds salt bridges
  • positively charged residues e.g., His, Arg, Lys
  • negatively charged residues e.g., Asp, Glu
  • disulfide bonds between nearby cysteine residues.
  • the present Example represents a Phase I first-in-man study of IMMU-114, to evaluate subcutaneous (SQ) EVIMU-l 14 injection in patients with recurrent/relapsed B-cell NHL or CLL.
  • Table 2 shows the demographics of the treated patient population. Eligible patients had recurrent/relapsed NHL/CLL with at least one prior therapy, ECOG performance status ⁇ 3, normal baseline renal and liver function, with platelets > 50,000/mm 3 and ANC > 1000/mm 3 .
  • Table 2 Patient Demographics for Phase I Clinical Trial of SQ anti-HLA-DR
  • Dose-limiting toxicity was defined as including: Grade 4 (or Grade 3 > 7 days) hematologic toxicity, excluding lymphopenia; Grade 4 (or Grade 3 requiring hospitalization/dialysis) injection reaction, infection or tumor lysis syndrome; Grade 4 fatigue > 7 days; other Grade 3 or 4 toxicity (excluding nausea/vomiting, electrolyte abnormalities w/o clinical sequelae, or liver function abnormality resolved to Grade 1 within 3 days of maximal
  • Treatment response was assessed 4 weeks after cycle 2, then every 3 months until progression, using 2007 IWG- HL or 2008 IW-CLL criteria.
  • one DLBCL patient with unrelated pneumonia withdrew after one dose had a PR (60% shrinkage) with a confirmatory scan pending, one FL patient progressed after cycle 2, one FL patient achieved a PR (83%) shrinkage continuing now 10 months later), and one CLL patient had an unconfirmed PR during cycle 1 (WBC ⁇ 50%> baseline, progressing after 2 months).
  • one patient with SLL progressed after cycle 2 and the other 2 patients with SAEs were not assessed for treatment response.
  • FIG. 29 A representative example of solid tumor reduction is shown in FIG. 29.
  • Patient 181- 001 had extensive abdominopelvic lymphadenopathy, including target lesions shown above (arrows).
  • Comparison of post-treatment CT images (POST) with baseline CT images (PRE) shows reduction in target lesions 4 weeks after completing 2 cycles of treatment at dose level 1 (200 mg once-weekly).
  • Antibody serum levels were evaluated on injection days by ELISA. For dose level 1 (once-weekly), IMMU-114 levels were not detectable ( ⁇ 24 ng/ml).
  • IMMU-114 levels on the last weekl injection day increased on treatment weeks 1, 2 and 3 of cycles 1 and 2, with peak concentrations of about 40 to 50 ng/ml seen at week 3 with both dose levels.
  • Table 3 summarizes the results from five patients with objective evidence of treatment activity. Activity was seen in five of ten evaluable patients (50%), including one complete response (10%). The best response by subgroup is shown in Table 4.
  • IMMU-114 demonstrated activity in this population relapsed/refractory to rituximab-containing therapies, the presence of short responses in some patients suggests treatment should be maintained beyond 2 cycles. Thus, the dosing scheme is being amended to allow treatment cycles to be repeated until disease progression, to determine an appropriate dosing schedule for undertaking a phase II study. In total, five of ten (50%) of assessable patients (FL x 2, CLL x 2, DLBCL) had objective evidence of treatment response, with one complete response observed. Circulating leukemic cells in CLL patients decreased with treatment, while B-cell changes in NHL patients were modest. The excellent safety profile and apparent therapeutic effect of IMMU-114 supports its use in hematologic cancers of human patients.
  • the hybridoma cell clone producing the mAb mL243 was cultured in HSFM medium (Life Technologies, Inc,) supplemented with 10% FBS (Hy clone).
  • the genes encoding the VK (VK 1 BACK/ CK3 ') and VH (VHIBACK/VHIFOR) of mL243 were cloned by RT-PCR and the sequences were determined by DNA sequencing. Multiple independent clones were sequenced to eliminate possible errors resulting from the PCR reaction.
  • hL243VHA represents nt 23 to 197 of the HL243VH domain
  • hL243VHB represents the minus strand of the hL243VH domain complementary to nt 176 to 343.
  • the 3'-terminal sequences (22 nt residues) of hL243VHA and B are complementary to each other, as underlined in the above sequences.
  • the 3'-ends of hL243VHA and B anneal to form a short double stranded DNA flanked by the rest of the long oligonucleotides. Each annealed end serves as a primer for the replication of the single stranded DNA in a PCR reaction, resulting in a double strand DNA composed of the nt 23 to 343 of hL243VH.
  • This DNA was further amplified in the presence of a short oligonucleotide primer pair, hRS7VHBACK and hL243VHFOR, to form the full-length hL243VH. Because of the sequence identity between hRS7VH and hL243VH in this region, hRS7VHBACK, previously designed and used for hRS7 Ab, was used here.
  • hL243VHA and B were amplified in the presence of 10 ⁇ of lOx PCR Buffer (500 mM KCl, 100 mM TrisHCL buffer, pH 8.3, 15 mM MgCl 2 ), 2 ⁇ of hRS7VHB ACK and hL243VHFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT).
  • This reaction mixture was subjected to 3 cycle of PCR reaction consisting of denaturation at 94°C for 1 minute, annealing at 45°C for 1 minute, and polymerization at 72°C for 15 minutes, and followed by 27 cycles of PCR reaction consisting of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute.
  • Double-stranded PCR-amplified product for hL243VH was gel-purified, restriction-digested with Pstl and BstEII and cloned into the complementary Pstl/BstEII sites of the heavy chain staging vector, VHpBS4.
  • hL243 VKA 155-mer
  • hL243VKB 155-mer
  • hL243VKA and B were amplified by two short oligonucleotides hlmmu31 VKBACK and hlmmu31 VKFOR as described above.
  • hlmmuS 1 VKB ACK and hlmmuSl VKFOR were designed and used previously for a humanized anti-AFP Ab (Qu et al, Clin Cancer Res (1999) 5 395-31).
  • hL243VKA represents nt 21 to 175 of the hL243VD domain
  • hL243VKB represents the minus strand of the hL243VK domain complementary to nt 154 to 312
  • a final expression vector hL243pdHL2 was constructed by sequentially subcloning the Xbal-BamHI and XhoI/NotI fragments of 1IL243V K and V H , respectively, into pdHL2 as described previously (Losman et al Cancer, 80:266, 1997).
  • the genomic sequence of human ⁇ chain was replaced with that of ⁇ 4 chain, which was obtained by PCR amplification.
  • the template used was the genomic DNA extracted from ATCC CRL-11397 cell and the primer pair was P-SacII
  • Ser241Pro (based on Kabat numbering) was introduced into the hinge region of the ⁇ 4 sequence to avoid formation of half-molecules when the IgG4 Ab is expressed in mammalian cell cultures (Schuurman et al, Mol Immunol 38: 1, 2001).
  • the human ⁇ 4 hinge region sequence between Pstl and Stul restriction sites (56 bp) was replaced with a synthetic DNA fragment with substitution of the TCA codon for Ser241 to CCG for Pro.
  • the human ⁇ genomic sequence in hL243pdHL2 was substituted with the mutated ⁇ 4 sequence, resulting in the final expression vector, designated as hL243y4PpdHL2, for the IgG4 isotype hL243.
  • a competition cell-binding assay was carried out to assess the immunoreactivity of hL243y4P relative to the parent mL243.
  • a constant amount of 125 I-labeled murine L243 or hL243y4P (100,000 cpm, -10 ⁇ / ⁇ &) was incubated with human lymphoma cells (Raji, Daudi or Ramos) in the presence of varying concentrations (0.2-700 nM) of purified hL243y4P or murine L243 at 4°C for 1-2 h. Unbound Abs were removed by washing the cells in PBS. The radioactivity associated with cells was determined after washing. As shown in FIG.
  • the antigen-binding affinity constant of hL243y4P was determined by direct cell surface binding assay of the radiolabeled antibodies and Scatchard plot analysis. To measure specific cell surface antigen binding, two sets of cells were prepared and used in the binding assay to estimate the non-specific and total binding of radioactivities, respectively. The cells for non-specific binding were pre-incubated with excess amount of unlabled Ab to block all surface antigen sites prior to adding the radiolabeled antibody, while those for total binding were pre-incubated in PBS.
  • Daudi cells were incubated with serial dilutions of the antibodies hL243, hL243y4P, hA20 (as another positive control) and hMN14 (negative control) in the presence of human complement for 2 h. This was followed by the addition of resazurin to assess cell viability. Both untreated and maximum lysis controls were included. Fluorescence readings were obtained 5 hours after resazurin addition. The fluorescence level obtained is directly correlated to the amount of viable cells. Percent viable cells was calculated by the formula (Test- maximum lysis)/(untreated control - maximum lysis) x 100.
  • Daudi cells were incubated with hA20, hL243, hL243y4P and hMN-14 at 5 ⁇ . Effector cells were added at a ratio of 5 : 1. After 4 hours cell lysis was assayed by LDH release (FIG. 9A) and cell lysis (FIG. 9B).
  • a multiplex colonmetric assays utilizing both MTS bioreduction (for determination of the number of viable cells) and BrdU (for quantification of cell proliferation based on the measurement of BrdU incorporation during DNA synthesis) were performed. Daudi and Raji cells were incubated with serial dilutions of hL243y4P for 2 and 3 days. mL243 and hMN-14 were used as positive and negative controls respectively. After incubation, BrdU and MTS assays were performed. Results of the MTS assays are shown in FIG. 10 and FIG. 11.
  • BRDU assays gave similar results (not shown). These results indicate hL243y4P inhibits proliferation of Raji and Daudi cell lines. However in similar experiments in the EBV negative cell line Ramos, inhibition of proliferation was only observed in the presence of a crosslinking anti Fc (Fab) 2 .
  • Fab crosslinking anti Fc
  • hL243 IgG4 isotype
  • lymphoma cell lines and a dog B cell lymphoma aspirate were tested for reactivity with lymphoma cell lines and a dog B cell lymphoma aspirate in comparison to the murine L243 as well as in comparison to other anti-B cell MAbs.
  • Two functional studies were also done.
  • the ability of the hL243 to induce apoptosis in the dog lymphoma aspirate was determined, and the antiproliferative activity of the hL243 was tested against Namalwa, a human lymphoma cell line reported to be resistant to rituximab.
  • the cell lines chosen were Namalwa (a rituximab resistant human B cell lymphoma cell line), SU-DHL-6 (a human B cell non-Hodgkin's lymphoma), WSU-FSCCL (an EBV-negative low-grade human B cell lymphoma cell line), Raji, Daudi, and Ramos cells.
  • Namalwa a rituximab resistant human B cell lymphoma cell line
  • SU-DHL-6 a human B cell non-Hodgkin's lymphoma
  • WSU-FSCCL an EBV-negative low-grade human B cell lymphoma cell line
  • Raji Daudi
  • Ramos cells Ramos cells.
  • hL243 bound to all the aforementioned cell lines.
  • hL243 demonstrated anti-proliferative activity in the rituximab resistant human B cell lymphoma cell line, Namalwa, as measured by a 3 H-thymidine uptake assay.
  • the hL243 MAb yielded 28% inhibition of proliferation when given alone. This was increased to 51% when hL243 was given in combination with anti-human IgG second antibody. When used in combination with rituximab the activity was increased to a level greater than that of either MAb alone. See FIG. 14-B.
  • anti-HLA- DR antibodies used in combination with other anti-TAA antibodies may exhibit synergistic effects against lymphoma and other diseases.
  • Phenotyping cell lines (Binding of humanized or chimeric MAbs on B cell lines by FACS Assay)
  • hL243 has a greater binding affinity to the dog lymphoma cells than other humanized MAbs. See Table 7. In addition, hL243 was able to induce apoptosis in the dog lymphoma cells when crosslinked with an anti-human IgG second antibody, measured as % cells with a sub GO/G1 phase DNA content (see FIG. 13). Table 7. Phenotyping dog lymphoma aspirate
  • the hybridoma cell clone producing the anti-HLA-DR monoclonal antibody, mL243 was obtained from ATCC (ATCC number HB-55). Cells were cultured in HSFM medium (Life Technologies, Inc ) supplemented with 10% FBS (Hy clone). The genes encoding the VK and V H genes of L243 were cloned by RT-PCR.
  • the IgG4/K isotype of hL243, hL243y4p can be constructed by replacing the heavy chain constant region coding sequence for the human ⁇ chain with that of ⁇ 4 chain.
  • a point mutation, Ser241Pro (based on Kabat numbering) was introduced into the hinge region of the ⁇ 4 sequence in order to avoid formation of half-molecules when the antibody is expressed and produced in mammalian cell cultures (Schuurman et al., Mol Immunol 38: 1 (2001)).
  • Other MAbs used in the studies were rituximab, purchased from IDEC Pharmaceuticals Corp.
  • hMN-14 used here as a negative isotype control, have been described previously (see, e.g., U.S. Patent No. 5,874,540).
  • Exemplary cell lines were used in several studies.
  • the Burkitt lymphoma lines, Daudi, Raji, and Ramos were purchased from the American Type Culture Collection (Manassas, VA).
  • Non-Burkitt lymphoma cell lines were obtained as follows: RL and SU-DHL-6, which contain the chromosomal translocation t(14; 18), were obtained from Dr John Gribben (Dana-Farber Cancer Institute, Boston, MA) and Dr Alan Epstein
  • Cell lines SU-DHL-4, SU-DHL-1, and Karpas 422 were provided by Dr Myron Czuczman (Roswell Park Cancer Institute, Buffalo, NY), and WSU-FCCL and DoHH2 cell lines were obtained from Dr Mitchell Smith (Fox Chase Cancer Center, Philadelphia, PA).
  • the cells were grown as suspension cultures in DMEM (Life Technologies, Gaithersburg, MD), supplemented with lo% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 ⁇ g/ml), and L-glutamine (2 mM) (complete media).
  • Immunophenotyping Indirect immunofluorescence assays were performed with the panel of cell lines described above, using FITC-goat anti-human IgG (Tago, Inc.,
  • Burlingame, CA Burlingame, CA essentially as described previously, and analyzed by flow cytometry using a FACSCaliber (Becton Dickinson, San Jose, CA).
  • F(ab') 2 goat anti -human IgG Fcy-specific (Jackson Laboratories) was similarly added to the second well from each primary MAb, and the volume of the third set was equalized by addition of medium. Following a 48-h incubation (37°C, 5% C0 2 ), cells were transferred to test tubes, washed with PBS, and then resuspended in hypotonic propidium iodide solution (50 mg/ml propidium iodide in 0.1% sodium citrate, 0.1% Triton X-100). Samples were analyzed by flow cytometry using a FACSCaliber. Percent apoptotic cells was defined as the percent of cells with DNA staining before G0/G1 peak (hypodiploid).
  • Flow cytometry analysis was performed using indirect immunofluorescent staining to show that hL243y4P binds to a panel of cultured human B cell lymphomas. A comparison to other surface antigens is shown. As seen in Table 8, the hL243y4P MAb binds to all the tested cell lines. A stronger expression was observed on Daudi and Raji, but the level of fluorescence staining is strong on all the cell lines tested. Binding was compared to that of humanized MAbs against other B cell antigens (CD74, CD22, CD20), the murine-human chimeric anti-CD20 MAb rituximab, and a humanized anti-CEA MAb (negative control).
  • the staining with hL243y4P was markedly greater than that of CD22 and CD74 on all seven cell lines.
  • CD20 staining was more variable, as shown by reactivity with the humanized (hA20) and chimeric (rituximab) MAbs.
  • SU-DHL-6 has higher CD20 expression, Namalwa, and WSU-FSCCL lower CD20 expression, and RL approximately equal expression of both antigens.
  • Induction of ADCC was measured in Raji, Daudi, and SU-DHL-6 by calcein AM release.
  • the activity of hL243y4P was compared to that of the murine L243 and rituximab, as a positive control.
  • rituximab and the murine L243 induced significantly more cell lysis than the negative controls (no MAb and murine and humanized MN-14) and hL243y4P did not (FIG. 16).
  • hL243 The effect of hL243 on cellular proliferation was assessed using the 3 H-thymidine uptake assay on Raji, FSCCL, and Namalwa cells (FIG. 17B and Table 9).
  • the effect of hL243y4P was compared to that of rituximab and to rituximab combined with hL243y4P, in the presence or absence of a crosslinking anti Fc antibody.
  • FSCCL previously shown to be relatively insensitive to rituximab
  • hL243y4P yielded significantly greater inhibition of proliferation than rituximab.
  • Ramos hL243 and rituximab activity were similar, and the combination was more effective than either alone.
  • the combination may have a synergistic effect.
  • Cross-linking with an anti-human Fc antibody was required for significant anti- proliferative activity to be seen in Ramos.
  • hL243y4P yielded significantly greater inhibition of proliferation than rituximab and the combination of rituximab and hL243y4P yielded significantly more inhibition of proliferation than either MAb alone.
  • hL243 y4P-induced cell death was evaluated by measuring various markers of apoptosis. These included induction of DNA fragmentation, Annexin V/7-AAD staining, measurement of activated caspase-3, loss of mitochondrial membrane potential and activation of the AKT survival pathway.
  • DNA fragmentation was evaluated by flow cytometry in SU-DHL-6 and Namalwa.
  • Cells were cultured with the MAbs for 48 h with or without a second MAb for crosslinking, followed by DNA staining with propidium iodide.
  • Cells were analyzed by flow cytometry, and positive florescence below the Gl region represents DNA fragmentation and is a measure of apoptosis.
  • hL243 ⁇ 4 ⁇ Activity of hL243 ⁇ 4 ⁇ was compared to that of humanized MAbs against other B cell antigens, including anti-CD74 (hLLl), anti CD22 (hLL2, epratuzumab), anti-CD20 (hA20), as well as the murine-human chimeric MAb rituximab. Controls included no first MAb and the negative control humanized anti-CEA MAb, hMN-14. hL243 ⁇ 4 ⁇ induced apoptosis in both cell lines, at levels similar to or greater than the other anti-B cell MAbs (FIG. 18A and 21B)
  • a kit was used (eg the Guava NexinTM kit) to discriminate between apoptotic and nonapoptotic dead cells in Daudi cells.
  • the kit utilizes Annexin-V-PE to detect
  • 7-AAD is excluded from live, healthy cells and early apoptotic cells, but permeates late stage apoptotic and dead cells.
  • FIG. 18B the results of this study indicated that hL243y4P induced apoptosis similar to mL243 following both 4 h and 24 h treatment.
  • the anti-CD20 MAb did not induce measurable apoptosis in Daudi cells. Therefore, hypercrosslinking by a secondary agent, such as anti-human IgG or protein A may be used for induction of apoptosis by anti- CD20 MAbs in many cell lines including Daudi.
  • Crosslinking with a second antibody may not be needed, but can increase the effect in 2 of 6 cell lines evaluated, FSCCL and Namalwa.
  • the loss of mitochondrial membrane potential induced by hL243y4P was greater than that of the anti-CD20 MAb (hA20), without a crosslinking agent. With crosslinking the hA20 levels are increased to those of hL243y4P in 3 of the 6 cell lines (RL, SU-DHL-6, and Daudi).
  • Cleaved caspase-3 was also assayed in Daudi over a 2 day time course (FIG. 20A). The activity continues to increase for approximately 2 days of L243y4P incubation. Time points less than 1 h were not done. Table 10. Cleaved Caspase-3 assay
  • a therapeutic study was performed to compare the in vivo efficacy of hL243y4P and mL243 (IgG2a isotype) monoclonal antibodies, in a xenograft model of human non- Hodgkin' s lymphoma (Raji).
  • the aim of this study was to determine if hL243y4P can maintain significant antitumor efficacy in a xenograft model.
  • SCID mice were injected with 2.5 x 10 6 Raji cells. Therapy with hL243y4P or mL243 was initiated 1 day-post tumor cell administration. Results are shown in FIG. 21.
  • mice injected with saline or with non-specific control antibody, hMN14 had a median survival time (MST) of 17 days. All the groups of mice treated with either humanized or murine L243 had significantly improved life span compared to mice injected with saline or hMN14 (P ⁇ 0.0001). Treatment with various doses of hL243y4P resulted in a dose-response relationship, with mice receiving higher doses having better survival times. In the group of animals treated with various doses of mL243 IgG2a, the cure rate was in the range of 80-100 %.
  • pharmacokinetic data on L243 and IMMU-114 administration were collected in normal dogs, followed by a preliminary trial of L243 in dogs with advanced lymphoma or unresectable plasmacytoma.
  • L243 and IMMU-114 were observed to bind to normal canine lymphocytes and canine lymphoma cells.
  • murine L243 and IMMU-114 binding yielded a reduction in viable cell counts and induction of apoptosis in canine lymphoma cells.
  • canine serum or peripheral blood mononuclear cells L243, but not EVIMU-114, induced CDC and ADCC, respectively.
  • both anti-HLA-DR mAbs can be administered safely to dogs and bind to malignant cells.
  • Evidence of clinical activity (hematopoietic toxicity and tumor response) was observed in dogs with advanced- stage lymphoma following L243 immunotherapy.
  • replacing the Fc region of a humanized IgGl anti-HLA-DR mAb with the IgG4 isotype abrogated the effector cell functions of the antibody (ADCC and CDC), while the antigen-binding properties, antiproliferative capacity ⁇ in vitro and in vivo), and the ability to induce apoptosis concurrent with activation of the AKT survival pathway and other signaling pathway effects, were retained.
  • IMMU-114 is indistinguishable from the parental murine mAb and a humanized IgGl anti-HLA-DR mAb in assays dependent upon antigen recognition.
  • the abrogation of ADCC and CDC may be preferred for in vivo therapeutic use.
  • Antibodies The following mAbs were used for phenotyping: anti-CD3-FITC, anti- CD4-FITC, anti-CD8-PE, and B cell-PE, purchased from Serotec Ltd (Raleigh, NC), unlabeled anti-human CD22 (LL2) and anti -human CD74 (LL1), supplied by Immunomedics, Inc.
  • L243 and humanized mAbs were from Immunomedics, Inc.
  • Flow cytometry Peripheral blood lymphocyte subsets were determined using flow cytometry. The different leukocyte populations were identified by their distinctive position on forward and side scatter plots. The lymphocyte population was gated and 10,000 events were acquired for each antibody. All flow cytometry experiments were performed and analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA). The data were analyzed with CellQuest software. Immunostaining was performed according to the manufacturer's directions. Briefly, a 100- ⁇ 1 aliquot of whole blood in EDTA was incubated with either antibody or isotype control antibody for 15 min at room temperature. Red blood cells were lysed with 2 ml of F ACS lysing solution and incubated for 5 min.
  • the cells were washed in phosphate-buffered saline (PBS), pH 7.4.
  • PBS phosphate-buffered saline
  • the cell pellet was resuspended in PBS containing 20 mM glucose and 1% bovine serum albumin and immediately assayed by flow cytometry.
  • HLA-DR and CD20 antigen expression on normal and neoplastic cells were performed by indirect immunofluorescence assays using FITC-goat anti-mouse IgG (GAM, Invitrogen, Carlsbad, CA), as described previously (Stein et al., Blood, 2006, 108:2736-44).
  • Apoptosis was evaluated by flow cytometry. Briefly, cells were incubated with mAbs for 48 h with or without a second antibody for cross-linking, followed by DNA staining with propidium iodide. Samples were analyzed by flow cytometry using a FACSCalibur. Percentage of apoptotic cells was defined as the percentage of cells with DNA staining before G1/G0 peak (hypodiploid).
  • Standard 51 Cr release assays were used to measure ADCC and CDC. Briefly, for CDC a 1/8 final dilution of canine serum was used as the source of complement, followed by a 3-h incubation. Cells treated with 0.25% Triton X-100 were included as 100% lysis control, and cells treated with complement alone as 0% lysis. For ADCC, effectontarget cell ratios of approximately 50: 1 were used, and incubations were for 4 h. All assays were performed in triplicate.
  • EDTA ethylenediamine tetraacetic acid
  • Dogs with Lymphoma Dogs were enrolled in this study if they had histologic or cytologic confirmation of lymphoma or plasma cell neoplasia and had previously failed or were refractory to conventional cytotoxic chemotherapy or if the owner had declined other therapy. Chemotherapy was not administered concurrently or less than 3 weeks prior to treatment with HLA-DR mAb. Pretreatment evaluation for all tumor-bearing dogs included physical examination, complete blood cell count, biochemical profile, and urinalysis. Dogs were excluded if there was evidence of > grade 2 toxicity on screening studies. Lymph nodes or tumors were measured in 3 dimensions and tumor volume was calculated as the product of length, width, height, ⁇ /6.
  • Enzyme-linked immunoabsorbent assay (ELISA). L243 and IMMU-1 14 serum levels were measured by ELISA. Two ml of whole blood were collected pretreatment, at the end of the antibody infusion, 1 h after the end of the infusion and at 24 h. The samples were allowed to clot at room temperature for 30 min and the serum was separated and frozen at -80° C prior to analysis. The ELISA assays were performed in 96-well PVC microtiter plates.
  • crosslinked L243 yields a specific therapeutic effect on canine lymphoma aspirates, leading to a reduction in viable cell count and induction of apoptosis, as measured by DNA fragmentation.
  • L243 induces CDC and ADCC.
  • IMMU-1 14 humanized, engineered L243 binds to canine lymphoma cells (Table 12).
  • FMMU-1 14 induces apoptosis in the canine lymphoma cells when crosslinked with an anti-human IgG second antibody (FIG. 23A).
  • Dog 1 had 1000/ ⁇ 1 band neutrophils 4 h after the second infusion (normal range O-100/ ⁇ ); Dog 2 had 1300/ ⁇ 1 band neutrophils 24 h after the first infusion. Both dogs had normal band neutrophil counts 24 h later. Lymphopenia (800/ ⁇ 1 - dog 1, 500/ ⁇ - dog 2: normal range 1000 - 4000/ ⁇ ) was noted 4-24 h following the first infusion in both dogs and following the second infusion in dog 2. Lymphocytes returned to normal within
  • Peripheral blood lymphocyte subset phenotyping indicated a decrease in both B and T cell lymphocytes (FIG. 24).
  • B and T cell lymphocytes Such rapid changes in neutrophils and lymphocytes represent a non-specific component to immunogens in dogs. Resolution of the neutrophilia occurred within one day and lymphocyte populations recovered over a 7-day period. Complete necropsy examination of Dogs 1 and 2 did not reveal any gross or histologic abnormalities.
  • Toxicity Infusional side-effects were common with 6/7 patients, experiencing grade 1 nausea or vomiting and 5/7 experiencing grade 1 fever. Slowing the infusion rate abrogated the adverse reactions.
  • Bone marrow aspirates indicated a non-specific granulocytic and megakaryocyte hypoplasia.
  • One dog was euthanized due to hemorrhage from multiple ulcerated cutaneous lymphoma lesions.
  • the second dog's cytopenias resolved uneventfully by the fourth week post infusion.
  • One dog died suddenly at home approximately 5 days after L243 therapy due to rapidly progressing, resistant lymphoma. A necropsy was not performed.
  • a comparison of cells aspirated from a lymph node prior to L243 with cells obtained one week after the first L243 infusion was performed in order to assess in vivo targeting of the L243 mAb.
  • the histograms represented baseline and one-week post infusion aspirated cells, to which no first or second antibodies were added in vitro (not shown).
  • the cells were incubated in vitro with FITC-labeled GAM, to detect cells that were labeled with L243 in vivo (not shown).
  • Cells obtained from the same lymph node 1 week after treatment with L243 were shifted to the right of the baseline cells, demonstrating the binding of murine IgG to the cell surface (not shown).
  • the cells were incubated in vitro with L243 and FITC-GAM to determine whether the cells were saturated with mAb L243.
  • Aspirated cells taken 1 week after treatment with L243 coincided with the baseline cells because the in vivo and in vitro binding of L243 IgG to the cell surface are indistinguishable after saturating doses of L243 (not shown).
  • Both groups exhibited higher mean fluorescence compared to that of the FITC-GAM labeled cells, indicating that the in vivo L243 dose administered did not saturate all malignant cells in the node (not shown).
  • Data obtained from cells aspirated 2 weeks after infusion continue to demonstrated L243 binding to malignant lymphocytes (not shown).
  • the L243 antibody was measured by ELISA in the serum of the last treated dog (152616). Samples were collected prior to the antibody infusion, at the end of the infusion, 1 h post infusion and at 24 h at each of the 4 treatments (FIG. 25). The serum level of L243 detected after the second infusion was markedly higher than after the first infusion. This suggests that the antigen pool present on cell surfaces was either blocked or eliminated by the first infusion. Infusions 3 and 4 yielded progressively lower serum concentrations of L243. This was likely due to an anti-antibody response causing rapid clearance of the infused murine L243 antibody. Because the presence of anti-mouse IgG was not measured, reappearance of an antigen sink cannot be ruled out.
  • IMMU- 1 14 administration in vivo Once F MU-l 14, the humanized reengineered IgG4 form of murine L243, became available, it was administered to 2 normal beagles at 3 mg/kg over 90 min. There was no infusion reaction noted in either dog during the infusion. One of the dogs was infused a second time 2 weeks later (at 1.3 mg/kg). A mild infusion reaction that included head shaking, mild fever and vomiting occurred following the second infusion. The severity of the reaction was lessened by slowing the rate of the infusion. This may suggest the development of anti-human IMMU-114 antibody.
  • CBCs and biochemical panels were conducted with no significant changes noted over a 2-week period, with the exception of a transient lymphopenia as also observed with L243 infusion.
  • Pharmacokinetic (PK) data obtained at the end of infusion, and 1, 4, 24, 48, 72 h, 1 week, and 2 weeks post- infusion indicated a rapid clearance within the first few hours, with about 50% of the IMMU- 114 antibody cleared within 2 h, and with the remaining antibody clearing with a half-life of ⁇ 2 days (FIG. 25).
  • Naturally-occurring lymphoma in dogs is extremely common and has been validated as a useful model of high-grade, B cell, non-Hodgkin's lymphoma in humans.
  • Conventional chemotherapeutic management of lymphoma in dogs, as in humans, is limited with 5-20% 2- year survival rates following CHOP -based chemotherapeutic protocols.
  • the availability of canine lymphoma patients, the ability to investigate novel strategies with repeated sampling of normal and tumor tissue or fluid, as well as the design of rigorous clinical trials to determine relevant therapeutic endpoints, are recognized advantages of this model as a bridge from preclinical investigations to humans.
  • anti-CD20 antibodies have contributed to improved outcomes in some forms of lymphoma in humans, the commercially available human anti-CD20 antibodies do not bind sufficiently with canine B cell lymphomas to permit further investigations of this strategy. However, substantial opportunities exist to expand the investigation of other antibody -based immunologic therapeutics.
  • Lymphoma is an increasingly common form of cancer with a wide range of immunologic and genetic subcategories with equally diverse prognoses. Aggressive forms of non-Hodgkin's lymphoma are currently controlled with chemotherapy with or without antibody infusions with only a moderate degree of success. Novel immunotherapeutic approaches, such as infusion of anti-B cell mAbs to improve the management of lymphoma, are traditionally examined in murine models but should be more carefully evaluated prior to human study to identify and better anticipate the impact of such interventions.
  • HLA-DR major histocompatibilty complex
  • B lymphocytes differentiation, and immunoglobulin secretion by B lymphocytes, as well as production of cytokines, modulation of expression of growth factor receptors, cell adhesion, and co- stimulatory molecules by B cells and monocytes (Nagy et al., J Mol Med, 2003, 81 :757-65).
  • HLA molecules have also been shown to serve as receptors that activate various cell death pathways, including caspase-dependent and caspase-independent alternative pathways of apoptosis (Nagy et al., J Mol Med, 2003, 81 :757-65; Mone et al., Blood, 2004, 103 : 1846-54; Newell et al., PNAS USA 1993, 90: 10459-63; Truman et al., Blood, 1997, 89: 1996-2007).
  • Functions reported to be affected by incubation of cells with L243 have included signal transduction, growth inhibition, Fas-mediated apoptosis, interactions with actin
  • Anti-HLA- DR (L243) was positive in 32/35 samples (greater than 5 units above the isotype control) and strongly positive (greater than 10 units above the negative control) in 30/35 samples. In contrast, anti-CD20 (2B8 used in these studies) was positive in 5/21, including 3 strongly positive. Reactivity of L243 was confirmed on the peripheral blood of several of these dogs.
  • Table 13 shows the relative expression of HLA-DR compared with CD74, CD22 and CD20 in different tumor types.
  • Table 15 illustrates the relative cytotoxicity of hL243y4P compared to other anti-B cell mAbs in different tumor types. The percent of untreated values in MTT assay are shown. Highlited values represent a significant decrease from untreated (P ⁇ 0.05). HLA-DR is expressed on all B-lymphoma and leukemia tested cell lines at markedly higher levels than CD20, CD22, and CD74. Despite positive staining AML cell lines are not killed by hL243g4P. Variation in expression and cytotoxicity profiles between the mAbs suggests that combination therapies may yield greater effects than the mAbs given singly.
  • HLA-DR HLA-DR
  • CD74 CD22
  • CD20 Cell line mAb control
  • FIG. 27 illustrates the ex vivo effects of various antibodies on whole blood.
  • hL243y4P resulted in significantly less B cell depletion than rituximab and veltuzumab, consistent with an earlier report (Nagy, et al, J Mol Med 2003;81 :757-65) which suggested that anti-HLA-DR mAbs kill activated, but not resting normal B cells, in addition to tumor cells. This suggests a dual requirement for both MHC-II expression and cell activation for antibody-induced death, and implies that because the majority of peripheral B cells are resting, the potential side effect due to killing of normal B cells may be minimal. T-cells are unaffected.
  • hL243y4P cytotoxicity correlates with activation of ERK and JNK signaling and differentiates the mechanism of action of hL243y4P cytotoxicity from that of anti-CD20 mAbs.
  • hL243y4P also changes mitochondrial membrane potential and generates ROS in Raji cells (not shown). Inhibition of ERK, INK, or ROS by specific inhibitors partially abrogates the apoptosis. Inhibition of 2 or more pathways abolishes the apoptosis.
  • hL243g4P may be useful in the treatment of mantle cell lymphoma, ALL, hairy cell leukemia, and CLL, as well as NHL and multiple myeloma.
  • the hL243 anti-HLA-DR antibody was designed, constructed, cloned and transfected into myeloma host cells as described in U.S. Patent No. 7,612, 180, the Examples section of which is incorporated herein by reference.
  • the purification process for hL243 IgG featured chromatography on three sequential columns of Protein A, Q-SEPHAROSE® and SP-SEPHAROSE®.
  • SEPHAROSE® is used as an exemplary column chromatography resin, the skilled artisan will realize that alternative methods of chromatography and alternative chromatography resins are known in the art and may be used. Further, the anion and cation exchange steps are not limited to Q-SEPHAROSE® and SP-SEPHAROSE®, but may also utilize other anion- and cation-exchange resins known in the art. The last step of the process utilizes a DV20 virus removal filtration, after which the product is tested for sterility.
  • the resin was packed up to a 20 cm height in a 20 cm diameter column to a packed bed volume of 6.3 L, with a maximum loading capacity of 220 gm.
  • the packed column was sanitized with 0.1 M acetic acid in 20% ethanol and then regenerated with 0.04 M PBS, pH 7.4. After equilibration, the supernatant was loaded at a maximum flow rate of 300 cm/hr. The column was washed with 0.04 M PBS, pH 7.4, until the absorbance returned to baseline, followed by washing with another 5 bed volumes of 0.04 M PBS, pH 7.4 at 300 cm/hr.
  • the bound IgG was eluted with 0.1 M citrate, pH 3.5, at a maximum flow rate of 300 cm/hr.
  • the elution profile was monitored by absorbance at 280 nm, using a flow through spectrophotometer.
  • the collected product peak was neutralized to pH 7.0 - 8.0 using 3 M Tris/HCl, pH 8.6.
  • the neutralized product peak was titrated to pH 3.5 - 3.7 using 1 M citric acid. This mixture was incubated at room
  • the diafiltered Protein A purified hLL2 IgG was filtered through a 0.2 ⁇ filter and stored at 2 - 8° C, before loading onto the Q-SEPHAROSE® column.
  • the anion exchange resin used for the next column was Q-SEPHAROSE® fast flow resin (GE Healthcare, Piscataway, NJ).
  • the resin was packed up to a 20 cm height in a 30 cm diameter column, to a packed bed volume of 14.1 L with a maximum loading capacity of 300 gm.
  • the packed column was sanitized with 1 M sodium hydroxide and then regenerated with 0.02 M Tris/HCl, 1.0 M NaCl, pH 8.0.
  • the resin was then equilibrated with 0.02 M Tris/HCl, 0.05 M NaCl, pH 7.5.
  • the diafiltered Protein A purified IgG was loaded at a flow rate of 100 cm/hr and the flow through peak was eluted with 0.02 M Tris/HCl, 0.05 M NaCl, pH 7.5 at a maximum flow rate of 300 cm/hr.
  • the contaminants eluted from the Protein A column bound to the Q-SEPHAROSE® resin.
  • the Q-SEPHAROSE® purified IgG was filtered using a 0.2- ⁇ filter and stored at 2-8°C until further purification. Before loading onto the final column, the IgG was titrated to pH 5.0 using 1 M citric acid.
  • the cation exchange resin used for the last column was SP-SEPHAROSE® fast flow resin (GE Healthcare, Piscataway, NJ). The resin was packed up to a 20 cm height in a 20 cm diameter column, with a maximum loading capacity of 220 gm. Before the Q- SEPHAROSE® purified hLL2 IgG was loaded, the packed column was sanitized with 1 M sodium hydroxide and then equilibrated with 0.025 M citrate, pH 5.0. The IgG was loaded at a maximum flow rate of 300 cm/hr and the column was washed with 5 bed volumes of 0.025 M citrate, pH 5.0, at 300 cm/hr. After loading and washing, the IgG was eluted with 0.025 M citrate, 0.15 M NaCl, pH 6.0. The elution profile was monitored by absorbance at 280 nm.
  • the purified hL243 IgG was concentrated to 10-11 mg/mL and diafiltered into 0.04 M PBS, pH 7.4, then filtered through 0.2 and 0.1 ⁇ filters before DV 20 filtration. After filtration, 75 mL of 0.04 M PBS, 1% Polysorbate 80, pH 7.4 was added to every liter of purified IgG and the mixture was filtered again through a 0.2 ⁇ filter before storage at 2°-8° C.
  • Exemplary antibodies tested include milatuzumab (hLLl, anti-CD74), epratuzumab (hLL2, anti-CD22), veltuzumab (hA20, anti-CD20) and hL243 (anti-HLA-DR; IMMU-114).
  • HCF High Concentration Formulation
  • this SQ formulation contains mannitol which has been of use in protein formulations for maintaining stability and isotonicity, and Polysorbate 80 (PS-80) which protects antibodies against aggregation. Since the pi value of most humanized IgGl antibodies is between 8 - 9.5, a citric acid/sodium citrate buffer system (buffering range 2.5 ⁇ 5.6) and a low pH (5.2) were used to ensure the protein is in charged form, and thus more stable in solution.
  • Polysorbate 80 1.0 mL (polysobate-80 was added at the end of the concentration (w/v) step)
  • HCF buffer The solute concentrations of HCF buffer were 6.2 mM citric acid monohydrate, 105 mM sodium chloride, 1.2 mM sodium citrate dihydrate, 8.7 mM sodium phosphate dibasic, 5.5 mM sodium phosphate monobasic, 66 mM mannitol, pH 5.2, conductivity 11.0 - 14.0 mS/cm.
  • An AMICON® Model 8050 Stirred Ultrafiltration Cell (from MILLIPORE®, 50 mL max volume) was used with a 50 kD polyethersulfone filter NMWL (from MILLIPORE®, diameter 44.5 mm) to concentrate the antibodies. Ultra pure argon gas was used to pressurize the system.
  • IgG could be concentrated by ultrafiltration up to 213 mg/mL without any visible aggregation or precipitation.
  • Other quality aspects of the antibody such as molecular integrity, charge variation and solution pH were also maintained.
  • Alternative high concentration formulations for subcutaneous or intramuscular administration may comprise amino acids, such as arginine or glutamine.
  • amino acids such as arginine or glutamine.
  • Epratuzumab was applied to a 40 mL MAB SELECT® (Protein A) affinity
  • the final CPREM formulation contained 66 mM mannitol, 100 mM arginine, 100 mM glutamic acid, 144 mM Na, 100 mM CI, 7.3 mM citrate, 22 mM phosphate, pH 5.3.
  • a protein concentration of 2.56 mg/mL was measured by UV spectrophotometry at 280 nM (OD 280 ).
  • the 600 mL solution was concentrated 120-fold using a stir-cell concentrator with a 50 kDa MWCO membrane.
  • a protein concentration of 238 mg/mL in the 120X concentrate was measured by OD 28 o. There was no evident precipitation by visual inspection and an SE- HPLC trace, which was indistinguishable from that of the pre-concentration material, showed no evidence of aggregation (data not shown).
  • the 120-fold concentrate was separated into three aliquots.
  • CPRE buffer 100 mM arginine, 100 mM glutamic acid, 144 mM Na, 100 mM CI, 7.3 mM citrate, 22 mM phosphate, pH 5.3.
  • the CPRE protein solution was concentrated until a precipitate was evident. At this point, concentration was terminated and the solution was filtered. The protein concentration in the filtered concentrate was measured at 99 mg/mL by OD 280 .
  • concentration in the filtered concentrate was measured at 137 mg/mL by OD 280 .
  • Veltuzumab was prepared for subcutaneous administration as described above.
  • veltuzumab injected s.c. every two weeks (Negrea et al., 201 1, Haematologica 96:567-573). Responses were assessed by CT scans, with other evaluations including adverse event, B-cell blood levels, serum veltuzumab levels and human anti-veltuzumab (HAHA) titers.

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Abstract

La présente invention concerne des compositions et des méthodes d'utilisation d'anticorps anti-HLA-DR ou de fragments de ceux-ci. Dans des modes de réalisation préférés, les anticorps sont administrés par voie sous-cutanée à un patient humain atteint d'un cancer hématologique ou d'une maladie auto-immune. Ledit anticorps anti-HLA-DR administré par voie sous-cutanée est efficace pour le traitement d'un cancer hématologique ou d'une maladie auto-immune chez des patients qui ont rechuté après des thérapies standards dirigées contre un cancer hématologique ou une maladie auto-immune, ou sont réfractaires auxdites thérapies, telles que l'administration d'anticorps anti-CD20, tels que le rituximab.
PCT/US2016/047483 2015-08-21 2016-08-18 Anticorps monoclonal anti-hla-dr à administrer par voie sous-cutanée pour le traitement de tumeurs malignes hématologiques WO2017034906A1 (fr)

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CA2987644A CA2987644A1 (fr) 2015-08-21 2016-08-18 Anticorps monoclonal anti-hla-dr a administrer par voie sous-cutanee pour le traitement de tumeurs malignes hematologiques
EP16839842.8A EP3337508A4 (fr) 2015-08-21 2016-08-18 Anticorps monoclonal anti-hla-dr à administrer par voie sous-cutanée pour le traitement de tumeurs malignes hématologiques

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US201562208128P 2015-08-21 2015-08-21
US62/208,128 2015-08-21
US14/876,200 2015-10-06
US14/876,200 US9683050B2 (en) 2011-05-02 2015-10-06 Stable compositions of high-concentration allotype-selected antibodies for small-volume administration
US201562262692P 2015-12-03 2015-12-03
US62/262,692 2015-12-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11780924B2 (en) 2016-06-21 2023-10-10 University Of Oslo HLA binding vaccine moieties and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112126A1 (en) * 1997-04-07 2005-05-26 Genentech, Inc. Anti-VEGF antibodies
US20100196267A1 (en) * 2005-03-03 2010-08-05 Immunomedics, Inc. Humanized L243 Antibodies

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2704751B1 (fr) * 2011-05-02 2019-04-17 Immunomedics, Inc. Concentration d'ultrafiltration d'anticorps à allotype sélectionné pour une administration de petit volume

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112126A1 (en) * 1997-04-07 2005-05-26 Genentech, Inc. Anti-VEGF antibodies
US20100196267A1 (en) * 2005-03-03 2010-08-05 Immunomedics, Inc. Humanized L243 Antibodies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIN, LY ET AL.: "An Antibody Treats Almost All Refractory Autoimmune Diseases: Fact and Beyond.", JOURNAL OF FORMOSAN MEDICAL ASSOCIATION., vol. 111, no. 4, April 2012 (2012-04-01), pages 181 - 182, XP055365426 *
See also references of EP3337508A4 *
STEIN, R ET AL.: "Therapy of B- cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, is Mediated Through Hyperactivation ot ERK and JNK MAPkinase Signaling Pathways.", BLOOD, vol. 115, no. 25, 24 June 2010 (2010-06-24), pages 5180 - 5190, XP055365427 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11780924B2 (en) 2016-06-21 2023-10-10 University Of Oslo HLA binding vaccine moieties and uses thereof

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CA2987644A1 (fr) 2017-03-02
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WO2017034906A8 (fr) 2017-10-12

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