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WO2003039486A2 - Anticorps anti-cd80 presentant une activite adcc visant la mort des cellules b de lymphome a mediation par adcc, isolement ou en combinaison avec d'autres therapies - Google Patents

Anticorps anti-cd80 presentant une activite adcc visant la mort des cellules b de lymphome a mediation par adcc, isolement ou en combinaison avec d'autres therapies Download PDF

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Publication number
WO2003039486A2
WO2003039486A2 PCT/US2002/036226 US0236226W WO03039486A2 WO 2003039486 A2 WO2003039486 A2 WO 2003039486A2 US 0236226 W US0236226 W US 0236226W WO 03039486 A2 WO03039486 A2 WO 03039486A2
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antibody
lymphoma
cell
antibodies
cells
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WO2003039486A3 (fr
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Kandasamy Hariharan
Nabil Hanna
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Idec Pharmaceuticals Corporation
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Publication of WO2003039486A3 publication Critical patent/WO2003039486A3/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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
    • 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
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention relates to the discovery that a PRI ATIZED ® IgGi antibody that shows specificity to the human CD80 molecule and which is referred to by the subject assignee, IDEC Pharmaceuticals Corporation, as IDEC-114 possesses antibody dependent cellular cytotoxicty (ADCC) against CD80 positive cells, especially CD80 positive cells of B cell lineage, and more particularly B cell lymphoma cells.
  • ADCC antibody dependent cellular cytotoxicty
  • the sequence of this PRIMATIZED ® antibody is disclosed in United States Patent No. 6,113,898 which is incorporated by reference in its entirety herein).
  • This primatized antibody is referred to as 16C10 therein).
  • the present invention also relates to the discovery that the use of IDEC-114 in combination with Rituxan ® , a chimeric anti-CD20 antibody approved by the FDA for treatment of non-Hodgkin's lymphoma, and/or chemotherapy yields a synergistic anti-tumor response against B cell lymphoma in vivo.
  • IDEC-114 or 16C10 as it is referred to in an earlier patent and applications by the inventors is a PRIMATIZED ® anti-CD80 lgd lambda monoclonal antibody (mAb) containing human constant regions and primate (cynomolgus macaque) variable regions.
  • mAb lgd lambda monoclonal antibody
  • This antibody binds specifically to human CD80 (B7.1), which is membrane-associated 60 KDa glycoprotein expressed an activated B cells, activated antigen presenting cells, and activated T cells.
  • Rituxmab is an unconjugated chimeric IgGi mAb that shows specificity to the human leukocyte antigen CD20. This antibody was developed at I DEC Pharmaceuticals Corporation and was approved for use by the Food and Drug Administration for treatment of relapsed or refractory, low-grade or follicular B-cell non-Hodgkin's lymphoma. (Rituxan ® Package Insert San Diego: IDEC Pharm. Corp., 4-19, 2001 , 2). The DNA and amino acid sequence for Rituxan ® are disclosed in U.S. Patent No. 5,736,137, which patent is incorporated by reference in its entirety herein. Additionally, a CHO cell transfectoma TCAE8 that expresses Rituxan has been deposited with the American Type Culture Collection (ATCC) and accorded ATCC deposit number 69119.
  • ATCC American Type Culture Collection
  • IDEC-114 is a PRIMATIZED ® antibody that shows specificity to the human CD80 molecule.
  • CD80 is expressed on activated B cells and on B-cell lymphoma.
  • anti-CD80 antibodies for treatment of lymphoma was within the scope of the invention disclosed therein.
  • This invention provides further information on this specific usage of anti-CD80 antibodies, and more particularly the primatized anti- CD80 antibodies which are the subject of U.S. Patent No. 6,113,898 and the divisional applications, thereof, which are incorporated by reference herein.
  • IDEC-114 alone or in combination with rituxmab (Rituxan ® ) exhibits an antitumor response against B-cell lymphoma in different experimental systems.
  • rituxmab rituxmab
  • ADCC Fc-dependent host effector cell-mediated cytotoxicity
  • IDEC-114 also exhibited complement-dependent cytotoxicity (CDC) with CD80 high expressing CHO cell transfectants, but failed to mediate CDC with CD80 + B-lymphoma cell lines expressing much lower levels of antigen.
  • CDC complement-dependent cytotoxicity
  • Fc-mediated effector mechanisms of IDEC-114 are more sensitive than complement when target cells contain limited amounts of antigen.
  • in vivo testing of IDEC-114 in a human B lymphoma/SCID mouse model demonstrated antitumor activity at 100, 200, and 400 ⁇ g per injection. The antitumor response observed with IDEC-114 was comparable to the antitumor response observed with rituximab at the same dose and treatment schedule.
  • IDEC-114 or another anti- CD80 antibody for treatment of B cell lymphoma or other B cell malignancies.
  • IDEC-114 in combination with an anti-CD20 antibody, preferably RITUXAN ® , for treatment of B cell lymphoma or other B cell malignancies.
  • an anti-CD20 antibody preferably RITUXAN ®
  • IDEC-114 or another anti-CD80 antibody in combination with a chemotherapeutic agent for treatment of B cell lymphoma or other B cell malignancies.
  • an anti-CD20 antibody preferably RITUXAN ®
  • an anti-CD80 antibody preferably IDEC-114.
  • Figure 1 depicts the pMS vector used to screen recombinant immunoglobulin libraries produced against B7 displayed on the surface of filamentous phage which contains primers based on macaque immunoglobulin sequences.
  • Figure 2 depicts the NEOSPLA expression vector used to express the subject primatized antibodies specific to human B7.1 antigen.
  • Figure 3 depicts monkey serum anti-B7.1 titers directed against cell surface B7.1 on transfected CHO cells.
  • Figure 4 depicts inhibition of radiolabeled sB7.1 binding by SB7.1 affinity- purified monkey antibodies in the presence of unlabeled SB7 and Mab L307.4 murine anti-B7.1.
  • Figure 5 depicts inhibition of binding of radiolabeled monkey 135 and L3707.4 anti-B7.1 antibodies to B7 positive human SB cells by competition with affinity-purified SB7.1.
  • Figure 6 depicts inhibition of radiolabeled B7-lg binding to activated human peripheral blood T cells by competing with unlabeled SB7.1 murine anti- B7.1 (L307.4) and monkey 1127 affinity purified serum antibodies.
  • Figure 7 depicts inhibition of IL-2 protein in mixed lymphocyte cultures by anti-B7.1 affinity-purified monkey serum antibodies.
  • Figure 8a depicts the amino acid and nucleic acid sequence of a primatized form of the light chain of 7C10.
  • Figure 8b depicts the amino acid and nucleic acid sequence of a primatized form of the heavy chain of 7C10.
  • Figure 9a depicts the amino acid and nucleic acid sequence of a primatized form of the light chain of 7B6.
  • Figure 9b depicts the amino acid and nucleic acid sequence of a primatized form of the heavy chain of 7B6.
  • Figure 10a depicts the amino acid and nucleic acid sequence of a primatized light chain 16C10.
  • Figure 10b depicts the amino acid and nucleic acid sequence of a primatized heavy chain 16C10.
  • Figure 11 compares the binding activity of two different lots of IDEC-114 to membrane bound CD80 cells determined by flow cytometry using CHO cells which express the CD80 molecule.
  • Figure 12 shows the ADCC activity of IDEC-114 and RITUXAN ® against SB cells and SKW cells by methods described in detail infra.
  • 51 Cr-labeled SB or SKW cells were incubated with varying concentration of IDEC-114, RITUXAN ® , human CE9-1 (an irrelevant isotype-matched control anti-human CD4 antibody) or murine (3C9) (an irrelevant isotype-matched control antibody) on activated human effector cells from peripheral blood at a 50:1 effector to target ratio.
  • 51 Cr released from target cells was measured and the percentage of specific lysis was then determined.
  • FIG 13 shows the ADCC activity of IDEC-114 and rituximab (RITUXAN ® ) combination.
  • the ADCC activity of IDEC-114 and rituximab in combination was determined on SKW cells as described infra. (Antibody-Dependent Cellular Cytotoxicity [ADCC]).
  • 51 Cr-labeled SKW cells were incubated with 10 ⁇ g/ml of IDEC-114/rituximab and activated host effector cells from peripheral blood of two donors (A and B) at 50:1 effector to target ratio. 51 Cr released from the target cells was measured and the percentage of specific lysis was determined.
  • Figure 14 shows the CDC activity of IDEC-114.
  • the CDC activity of IDEC-114 and rituximab was determined on CD80-expressing CHO (a), SKW (b), or Daudi (c) ells as described infra in the examples. (Complement-Dependent Cytotoxicity [CDC]).
  • 51 Cr-labeled target cells were incubated with IDEC-114, rituximab, or control antibodies at indicated antibody concentrations with and without complement. After 4 hours of incubation, 51 Cr released from the target cells was measured and the percentage of specific lysis was determined.
  • Figure 15 shows the antitumor response elicited by IDEC-114 in SKW/SCID mice.
  • groups of mice were inoculated intravenously with 3 x 10 6 SKW cells.
  • Figure 16 shows the antitumor response elicited by IDEC-114 and RITUXAN ® in SKW/SCID mice.
  • Mice in the combination treatment groups were injected intraperitonealy with 200 ⁇ g each of IDEC-114 and rituximab.
  • Mice in the monotherapy treatment groups received 200 ⁇ g or 400 ⁇ g of IDEC-114 or rituximab. All antibody injections were given in a final volume of 200 ⁇ l.
  • Mice in the control group were injected with a formulation buffer in a volume equal to the antibody injection volume.
  • Antibody injections were given on days 1 , 3, 5, 7, 9, and 11 after tumor inoculation. Mice were monitored for disease development and death.
  • Figure 17 is a schematic depiction of a neoplastic B cell bound by IDEC- 114 and RITUXAN ® .
  • Figure 18 is schematic depiction of a human B-cell lymphoma/SCID mouse model of disease-free survival and protection after antibody administration.
  • Figure 19 shows results of combination treatment with IDEC-114 and adriamycin in a human lymphoma/SCID mouse model.
  • anti-CD80 antibodies have been reported previously as a treatment modality for autoimmune diseases and transplantation.
  • the present invention is directed toward the use of an anti-CD80 antibody, preferably IDEC-114, for sole or combination therapy for the treatment of B cell lymphoma.
  • CD80 is a suitable potential target for lymphoma and other B cell malignancies as it is expressed on malignant B cells.
  • an anti-CD80 antibody has not previously been demonstrated to be suitable for treatment of B cell lymphoma.
  • CD80 is expressed on some malignant B cells, it was theorized that IDEC-114 would be an effective anti-tumor agent against B cell lymphoma cells. Moreover, because CD80 is not expressed in early hematopoietic stem ceils, it potentially is an attractive agent for B cell lymphoma therapy as it should not affect B cell and other immune cell proliferation and therefore should not necessitate bone marrow transplant after therapy.
  • the anti-CD20 antibody and/or the chemotherapeutic agent enhances the ADCC response against CD80 positive cells, e.g., B cell lymphoma cells that is elicited by the anti-CD80 antibody. Consequently, the present invention is directed in part to the combined use of an anti-CD80 antibody and an anti-CD20 antibody and/or a chemotherapeutic agent, for treating B cell lymphoma. Also, the invention is directed to the enhancement of the ADCC activity of an anti-CD80 antibody against CD80 positive cells by co- administering this antibody with an anti-CD20 antibody.
  • the anti-CD80 antibody will comprise IDEC-114 or an anti-CD80 antibody having one or more of the following properties:
  • the anti-CD80 antibody will be IDEC-114 or a comparable human, humanized or PRIMATIZED ® antibody.
  • the anti-CD20 antibody will comprise RITUXAN ® or will comprise an anti-CD20 antibody having one or more of the following properties:
  • B cell lymphoma cells as RITUXAN ® , i.e. at least 75% that RITUXAN ® , and more preferably at least 90% of RITUXAN ® ;
  • the anti-CD20 antibody will comprise RITUXAN ® , given its established clinical efficacy.
  • antibodies can be raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified tumor associated antigens or cells or cellular extracts comprising such antigens) and an adjuvant.
  • relevant antigen e.g., purified tumor associated antigens or cells or cellular extracts comprising such antigens
  • This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes. While the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood, to provide homogenous preparations of monoclonal antibodies (MAbs). Preferably, the lymphocytes are obtained from the spleen.
  • lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • an immortal tumor cell line e.g. a myeloma cell line
  • hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They therefore produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
  • Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
  • culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
  • the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro assay, such as a radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)).
  • the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • DNA encoding the desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • the isolated DNA (which may be modified as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No.. 5,658,570, filed January 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification thereof by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
  • DNA encoding other anti- CD80 and anti-CD20 antibodies or antibody fragments may also be derived from antibody phage libraries as set forth, for example, in EP 368 684 B1 and U.S.P.N. 5,969,108 each of which is incorporated herein by reference.
  • Several publications e.g., Marks et al. Bio/Technology 10:779-783 ( 992) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. Such procedures provide viable alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies and, as such, are clearly within the purview of the instant invention.
  • Yet other embodiments of the present invention comprise the generation of substantially human anti-CD20 or anti-CD80 antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference
  • IDEC-114 is a primatized anti-CD80 antibody, provided according to these references as are the other exemplified anti-CD80 antibodies disclosed in U.S. Patent No. 6,113,898. It will further be appreciated that the scope of this invention further encompasses all alleles, variants and mutations of the DNA sequences described herein.
  • RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligodT cellulose. Techniques suitable to these purposes are familiar in the art and are described in the foregoing references.
  • cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods. It may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
  • DNA typically plasmid DNA
  • DNA may be isolated from the cells as described herein, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail in the foregoing references relating to recombinant DNA techniques.
  • the DNA may be modified according to the present invention at any point during the isolation process or subsequent analysis.
  • Preferred antibody sequences are disclosed herein in the examples and in the IDEC patents incorporated by reference herein. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to the skilled artisan and may be carried out using any of several commercially available automated synthesizers. In addition, DNA sequences encoding several types of heavy and light chains set forth herein can be obtained through the services of commercial DNA synthesis vendors. The genetic material obtained using any of the foregoing methods may then be altered or modified to provide antibodies compatible with the present invention.
  • modified antibodies are held to mean immunoglobulins, antibodies, or immunoreactive fragments or recombinants thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as the ability to non-covalently dimerize, increased tumor localization or reduced serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
  • immunoreactive single chain antibody constructs having altered or omitted constant region domains may be considered to be modified antibodies.
  • immunoglobulin comprises five distinct classes of antibody that can be distinguished biochemically. While all five classes are clearly within the scope of the present invention, the following discussion will generally be directed to the class of IgG molecules.
  • immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” and continuing through the variable region.
  • both the light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used functionally.
  • the variable domains of both the light (V ⁇ _) and heavy (VH) chains determine antigen recognition and specificity.
  • the constant domains of the light chain (C ) and the heavy chain (CH1 , CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody.
  • the CH3 and C L domains actually comprise the carboxy-terminus of the heavy and light chains respectively.
  • Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunogobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. At the N-terminus is a variable region and at the C- terminus is a constant region.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them. It is the nature of this chain that determines the "class" of the antibody as IgA, IgD, IgE IgG, or IgM.
  • the immunoglobulin subclasses e.g. IgG-i, lgG 2 , lgG 3 , lgG 4 , IgAi, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discemable to the skilled artisan in view of the instant disclosure and, accordingly, are within the purview of the instant invention.
  • variable region may comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against CD80 or CD20.
  • the variable region of the modified antibodies may be, for example, of human, murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.) or lupine origin.
  • both the variable and constant regions of compatible modified antibodies are human.
  • the variable regions of compatible antibodies (usually derived from a non-human source) may be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule.
  • variable regions useful in the present invention may be humanized or otherwise altered through the inclusion of imported DNA or amino acid sequences.
  • humanized antibody shall mean an antibody derived from a non-human antibody, typically a murine antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues.
  • CDRs complementarity determining regions
  • chimeric antibodies will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species.
  • the antigen binding region or site will be from a non- human source (e.g. mouse) and the constant region is human. While the immunogenic specificity of the variable region is not generally affected by its source, a human constant region is less likely to elicit an immune response from a human subject than would the constant region from a non-human source.
  • variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. It must be emphasized that it may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site. Given the explanations set forth in U. S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.
  • modified antibodies useful in the instant invention will comprise antibodies, or immunoreactive fragments thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased tumor localization or reduced serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region.
  • the constant region of the modified antibodies will comprise a human constant region, preferably IgGi.
  • Modifications to the constant region compatible with the instant invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise alterations or modfications to one or more of the three heavy chain constant domains (CH1 , CH2 or C ⁇ 3) and/or to the light chain constant domain (CL).
  • the C 2 domain of a human IgG Fc region usually extends from about residue 231 to residue 340 using conventional numbering schemes.
  • the C ⁇ 2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule.
  • the CH3 domain extends from the CH2 domain to the C-terminal of he IgG molecule and comprises approximately 108 residues while the hinge region of an IgG molecule joins the CH2 domain with the CH1 domain. This hinge region encompasses on the order of 25 residues and is flexible, thereby allowing the two N-terminal antigen binding regions to move independently.
  • the antibody constant region mediates several effector functions. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • FcR Fc receptor
  • Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell- mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell.
  • vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • vector systems may be employed to produce anti-CD80 and/or anti-CD20 antibodies useful in the subject sole and combination B cell malignancy therapies.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • biocide resistance e.g., antibiotics
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) modified as discussed above.
  • this is effected using a proprietary expression vector of IDEC, Inc., referred to as NEOSPLA.
  • This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
  • this vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
  • This vector system is substantially disclosed in commonly assigned U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, i.e., > 30 pg/cell/day.
  • the modified antibodies of the instant invention may be expressed using polycistronic constructs such as those disclosed in copending United States provisional application No. 60/331 ,481 filed November 16, 2001 and incorporated herein in its entirety.
  • polycistronic constructs such as those disclosed in copending United States provisional application No. 60/331 ,481 filed November 16, 2001 and incorporated herein in its entirety.
  • multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
  • These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of modified antibodies in eukaryotic host cells.
  • IRES sequences are disclosed in U.S.P.N. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of modified antibodies disclosed in the instant application.
  • the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988).
  • plasmid introduction into the host is via electroporation.
  • the transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or flourescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • transformation shall be used in a broad sense to refer to any introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
  • host cells refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • antibodies or modifications thereof produced by a host cell that is, by virtue of this transformation, recombinant.
  • the terms "cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of antibody from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • the host cell line used for antibody expression is most preferably of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney).
  • DG44 and DUXB11 Choinese Hamster Ovary lines,
  • CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature. [0072] In vitro production allows scale-up to give large amounts of the desired antibody. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. As previously described, at least some of the monomeric subunits spontaneously associate non-covalently to form dimeric antibodies.
  • the immunoglobulins in the culture supernatants may first be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as PEG, filtration through selective membranes, or the like. If necessary and/or desired, the concentrated solutions of tetravalent antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography.
  • Antibody genes can also be expressed non-mammalian cells such as bacteria or yeast.
  • various unicellular non- mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation.
  • Bacteria which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae.
  • the immunoglobulin heavy chains and light chains typically become part of inclusion bodies. The chains then must be isolated, purified and then assembled into functional antibodies.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. [0075] For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used.
  • This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • the anti-CD80 or anti-CD20 antibodies used in the therapeutic methods of the present invention may be used in any one of a number of conjugated (i.e. an immunoconjugate) or unconjugated forms.
  • unconjugated antibodies are generally preferred given the pronounced ADCC activity of IDEC-114 and RITUXMAN ® , which are the preferred embodiments of the invention.
  • the antibodies of the present invention may be conjugated to cytotoxins such as radioisotopes, therapeutic agents, cytostatic agents, biological toxins or prodrugs.
  • the dimeric antibodies of the instant invention may be used in a nonconjugated or original form to harness the subject's natural defense mechanisms to eliminate the malignant cells.
  • the antibodies may be conjugated to radioisotopes, such as 90 Y, 25 l, 131 l, 123 l, 111 ln, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re using anyone of a number of well known chelators or direct labeling.
  • the disclosed compositions may comprise antibodies coupled to drugs, prodrugs or biological response modifiers such as methotrexate, adriamycin, and lymphokines such as interferon.
  • Still other embodiments of the present invention comprise the use of antibodies conjugated to specific biotoxins such as ricin or diptheria toxin.
  • the modified antibodies may be complexed with other immunologically active ligands (e.g. antibodies or fragments thereof) wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell.
  • immunologically active ligands e.g. antibodies or fragments thereof
  • the selection of which conjugated or unconjugated modified antibody to use will depend of the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
  • a cytotoxin or cytotoxic agent means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or distroy a cell or malignancy when exposed thereto.
  • exemplary cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines.
  • radionuclide cytotoxins are particularly preferred for use in the instant invention.
  • any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells or to eliminate these cells and may be associated with the antibodies disclosed herein is within the purview of the present invention.
  • radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy ⁇ - or ⁇ -particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy ⁇ -, ⁇ - or ⁇ -particles which have a therapeutically effective path length.
  • Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They generally have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • the antibodies may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent.
  • a chelating agent is covalently attached to an antibody and at least one radionuclide is associated with the chelating agent.
  • Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope.
  • Particularly preferred chelating agents comprise 1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid (“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives.
  • Other chelating agents comprise P-DOTA and EDTA derivatives.
  • Particularly preferred radionuclides for indirect labeling include 111 ln and 90 Y.
  • direct labeling and “direct labeling approach” both mean that a radionuclide is covalently attached directly to a dimeric antibody (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the antibody, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention.
  • Technetium-99m labelled antibodies may be prepared by ligand exchange processes, by reducing pertechnate (TcO 4 " ) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the antibodies to this column, or by batch labelling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCI 2 , a buffer solution such as a sodium- potassium phthalate-solution, and the antibodies.
  • a reducing agent such as SnCI 2
  • a buffer solution such as a sodium- potassium phthalate-solution
  • Modified antibodies according to the invention may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidising agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidising agent, such as lactoperoxidase, glucose oxidase and glucose.
  • a chemical oxidising agent such as sodium hypochlorite, chloramine T or the like
  • an enzymatic oxidising agent such as lactoperoxidase, glucose oxidase and glucose.
  • the indirect labeling approach is particularly preferred.
  • Patents relating to chelators and chelator conjugates are known in the art.
  • U.S. Patent No. 4,831 ,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation.
  • U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, 5,434,287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety.
  • compatible metal chelators are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1 ,4,8,11-tetraazatetradecane, 1 ,4,8,11-tetraazatetradecane-1 ,4,8,11- tetraacetic acid, 1-oxa-4,7,12,15-tetraazaheptadecane-4,7,12,15-tetraacetic acid, or the like. Cyclohexyl-DTPA or CHX-DTPA is particularly preferred and is exemplified extensively below. Still other compatible chelators, including those yet to be discovered, may easily be discerned by a skilled artisan and are clearly within the scope of the present invention.
  • Compatible chelators including the specific bifunctional chelator used to facilitate chelation in co-pending application Serial Nos. 08/475,813, 08/475,815 and 08/478,967, incorporated by reference in their entirety herein, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non- tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites.
  • target sites i.e., B-cell lymphoma tumor sites.
  • other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
  • antibodies may be conjugated to different radiolabels for diagnostic and therapeutic purposes.
  • radiolabeled therapeutic conjugates for diagnostic "imaging" of tumors before administration of therapeutic antibody.
  • "ln2B8" conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 111 ln via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1 :1 mixture of 1-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl-3- isothiocyanatobenzyl-DTPA.
  • MX-DTPA diethylenetriaminepentaacetic acid
  • 111 ln is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90 Y-labeled antibody distribution.
  • 131 1 is a well known radionuclide used for targeted immunotherapy.
  • the clinical usefulness of 131 l can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor.
  • 90 Y provides several benefits for utilization in radioimmunotherapeutic applications: the 64 hour half-life of 90 Y is long enough to allow antibody accumulation by tumor and, unlike e.g., 131 l, 90 Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1 ,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90 Y-labeled antibodies. Additionally, internalization of labeled antibody is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target antigen.
  • Effective single treatment dosages (i.e., therapeutically effective amounts) of 90 Y-labeled modified antibodies range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi.
  • Effective single treatment non-marrow ablative dosages of 131 l-labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi.
  • Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 131 l-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi.
  • an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCi. Imaging criteria for, e.g., the 111 ln label, are typically less than about 5 mCi.
  • radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging.
  • additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 123 l, 125 l, 32 P, 57 Co, 64 Cu, 67 Cu, 77 Br, 81 Rb, 81 Kr, 87 Sr, 13 ln, 127 Cs, 129 Cs, 132 l, 19 Hg, 203 Pb, 206 Bi, 177 Lu, 186 Re, 212 Pb, 212 Bi, 47 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 199 Au, 225 Ac, 211 At, and 213 Bi.
  • radionuclides are compatible with a selected course of treatment without undue experimentation.
  • additional radionuclides which have already been used in clinical diagnosis include 125 l, 123 l, 99 Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, as well as 111 ln.
  • Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy Peirersz et al. Immunol. Cell Biol. 65: 111-125 (1987).
  • These radionuclides include 188 Re and 186 Re as well as 199 Au and 67 Cu to a lesser extent.
  • U.S. Patent No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
  • the antibodies of the present invention may be conjugated to, or associated with, any one of a number of biological response modifiers, pharmaceutical agents, toxins or immunologically active ligands.
  • these non-radioactive conjugates may be assembled using a variety of techniques depending on the selected cytotoxin.
  • conjugates with biotin are prepared e.g. by reacting the antibodies with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester.
  • conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed above, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate.
  • Conjugates of the antibodies of the invention with cytostatic/cytotoxic substances and metal chelates are prepared in an analogous manner.
  • Preferred agents for use in the present invention are cytotoxic drugs, particularly those which are used for cancer therapy.
  • Such drugs include, in general, cytostatic agents, alkylating agents, antimetabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like.
  • cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine.
  • cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6- mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Hormones and hormone antagonists such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g.
  • tamoxifen, androgens e.g. testosterone
  • aromatase inhibitors e.g. aminogluthetimide
  • one skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
  • compatible cytotoxins may comprise a prodrug.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5- fluorouridine prodrugs that can be converted to the more active cytotoxic free drug.
  • Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
  • antibodies can also be associated with a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • cytokines such as lymphokines and interferons.
  • radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy.
  • An antibody conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal.
  • the unbound radiosensitizer linked modified antibodies would be cleared quickly from the blood, localizing the remaining radiosensitization agent in the target tumor and providing minimal uptake in normal tissues.
  • adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting antibody.
  • a potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
  • preferred embodiments of the invention comprise the administration of an anti-CD80 antibody preferably one having ADCC activity, to a patient with a B cell malignancy, e.g., leukemia or lymphoma or in combination or conjunction with one or more other therapies such, in particular anti-CD20 antibody therapy, and/or chemotherapy or radiotherapy (i.e. a combined therapeutic regimen).
  • a B cell malignancy e.g., leukemia or lymphoma
  • one or more other therapies such, in particular anti-CD20 antibody therapy, and/or chemotherapy or radiotherapy (i.e. a combined therapeutic regimen).
  • the administration of antibodies in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the subject anti-CD80 and/or anti-CD20 antibodies.
  • chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by radioimmunoconjugates of the present invention.
  • cytotoxin associated antibodies could be administered intravenously followed by tumor localized external beam radiation.
  • the antibody may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit.
  • a skilled artisan e.g. an experienced oncologist
  • the combination of the anti-CD80 antibody (with or without cytotoxin) and a chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and anti-CD80 antibody may be administered in any order or concurrently.
  • the antibodies of the present invention will be administered to patients that have previously undergone chemotherapy.
  • the antibodies and the chemotherapeutic treatment will be administered substantially simultaneously or concurrently.
  • a B cell lymphoma patient may be given the anti-CD80 antibody while undergoing a course of chemotherapy.
  • the modified antibody will be administered within 1 year of any chemotherapeutic agent or treatment.
  • the ant-CD80 antibody will be administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment.
  • the dimeric antibody will be administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment.
  • the dimeric antibody will be administered within 5, 4, 3, 2 or 1 days of the selected chemotherapeutic agent or treatment. It will further be appreciated that the two agents or treatments may be administered to the patient within a matter of hours or minutes (i.e. substantially simultaneously).
  • the anti-CD80 antibodies used in the instant invention may be used in conjunction or combination with any chemotherapeutic agent or agents (e.g. to provide a combined therapeutic regimen) that eliminates, reduces, inhibits or controls the growth of neoplastic cells in vivo. As discussed, such agents often result in the reduction of red marrow B reserves.
  • the radiolabeled immunoconjugates disclosed herein may be effectively used with radiosensitizers that increase the susceptibility of the neoplastic cells to radionuclides.
  • radiosensitizing compounds may be administered after the radiolabeled modified antibody has been largely cleared from the bloodstream but still remains at therapeutically effective levels at the site of the tumor or tumors.
  • exemplary chemotherapic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone.
  • alkylating agents e.g., vincristine and vinblastine
  • procarbazine methotrexate
  • prednisone methotrexate
  • MOPP mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone
  • ABVD e.g., adriamycin, bleomycin, vinblastine and dacarbazine
  • ChlVPP chlorambucil, vinblastine, procarbazine and prednisone
  • CABS lomustine, doxorubicin, bleomycin and streptozotocin
  • MOPP plus ABVD MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations
  • Additional regimens that are useful in the context of the present invention include use of single alkylating agents such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine,
  • CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide.
  • Other compatible chemotherapeutic agents include, but are not limited to, 2- chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin and fludarabine.
  • Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, etoposide and ifosfamide given alone or in combination.
  • IMVP-16 ifosfamide, methotrexate and etoposide
  • MIME methyl-gag, ifosfamide, methotrexate and etoposide
  • DHAP dexamethasone, high dose cytarabine and cisplatin
  • ESHAP etoposide, methylpredisolone, HD cytarabine, cisplatin
  • CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin
  • CAMP lomustine, mitoxantrone, cytarabine and prednisone
  • chemotherapeutic agent used in combination with the antibodies of the instant invention may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et al., Antineoplastic Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman et al., eds., 9 th ed. 1996).
  • the antibodies of the present invention are administered in a pharmaceutically effective amount for the in vivo treatment of B cell malignancies, particularly leukemias and lymphomas.
  • the disclosed antibodies will be formulated so as to facilitate administration and promote stability of the active agent.
  • pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • a pharmaceutically effective amount of the dimeric antibody, immunoreactive fragment or recombinant thereof, conjugated or unconjugated to a therapeutic agent shall be held to mean an amount sufficient to achieve effective binding with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells.
  • the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the antibody.
  • the subject therapies should be useful for reducing tumor size, inhibiting tumor growth and/or prolonging the survival time of tumor- bearing animals.
  • this invention also relates to a method of treating tumors in a human or other animal by administering to such human or animal an effective, non-toxic amount of antibody.
  • an effective, non-toxic amount of antibody would be for the purpose of treating CD80 positive malignancies.
  • a therapeutically active amount of a antibody may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject.
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.
  • mammal refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment of a B cell malignancy e.g., B cell lymphoma, include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented.
  • the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease.
  • the CD80 and CD20 antibodies of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce such effect to a therapeutic or prophylactic degree.
  • the antibodies of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • a cocktail comprising one or more species of dimeric antibodies according to the present invention may prove to be particularly effective.
  • Methods of preparing and administering conjugates of the antibody, immunoreactive fragments or recombinants thereof, and a therapeutic agent are well known to or readily determined by those skilled in the art.
  • the route of administration of the antibody or antibodies (or fragment thereof) of the invention may be oral, parenteral, by inhalation or topical.
  • parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • a preferred administration form would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
  • a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumine), etc.
  • the dimeric antibodies can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased CD80 positive tissue to the therapeutic agent.
  • Preparations for parenteral administration includes sterile aqueous or non-tumor aqueous solutions, suspensions, and emulsions.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.
  • Intravenous vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating an active compound (e.g., a dimeric antibody by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • an active compound e.g., a dimeric antibody by itself or in combination with other active agents
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S.S.N. 09/259,337 and U.S.S.N. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to B cell neoplastic disorders.
  • preferred embodiments of the present invention provide compounds, compositions, kits and methods for the treatment of neoplastic B cell disorders in a mammalian subject in need of treatment thereof.
  • the subject is a human.
  • the B cell neoplastic disorder e.g., cancers and malignancies
  • the disclosed invention may be used to prophylactically or therapeutically treat any neoplasm comprising CD80 that allows for the targeting of the antibody to the cancerous cells.
  • Exemplary hematologic malignancies that are amenable to treatment according to the disclosed invention include Hodgkins and non-Hodgkins lymphoma as well as leukemias, including ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL) and monocytic cell leukemias.
  • ALL-L3 Breast Tumor's type leukemia
  • CLL chronic lymphocytic leukemia
  • monocytic cell leukemias monocytic cell leukemias.
  • the antibodies and compounds and methods of the present invention are particularly effective in treating a variety of B-cell lymphomas, including low grade/ follicular non- Hodgkin's lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's Macroglobulinemia.
  • NHL low grade/ follicular non- Hodgkin's lymphoma
  • FCC cell lymphoma
  • MCL mantle cell lymphoma
  • DLCL diffuse large cell lymphoma
  • SL small lymphocytic NHL
  • intermediate grade/ follicular NHL intermediate grade diffuse NHL
  • high grade immunoblastic NHL high grade lymphoblastic NHL
  • lymphomas will often have different names due to changing systems of classification, and that patients having lymphomas classified under different names may also benefit from the combined therapeutic regimens of the present invention.
  • the disclosed invention may advantageously be used to treat additional malignancies bearing compatible tumor associated antigens.
  • This vector, pMS, Figure 1 contains a single lac promoter/operator for efficient transcription and translation of polycistronic heavy and light chain monkey DNA.
  • This vector contains two different leader sequences, the omp A (Movva et al, J. Biol. Chem., 255: 27-29, (1980), for the light chain and the pel B (Lei, J. Bact. 4379-109:4383 (1987) for the heavy chain Fd. Both leader sequences are translated into hydrophobic signal peptides that direct the secretion of the heavy and light chain cloned products into the periplasmic space. In the oxidative environment of the periplasm, the two chains fold and disulfide bonds form to create stable Fab fragments.
  • helper phage The replication and assembly of pMS DNA strands into phage particles requires viral proteins that must be provided by a helper phage.
  • helper phage VCSM13 which is particularly suited for this, since it also contains a gene coding for kanamycin resistance.
  • Bacteria infected with VCSM13 and pMS can be selected by adding both kanamycin and carbenicillin to the growth medium. The bacteria will ultimately produce filamentous phage particles containing either pMS or VCSM13 genomes.
  • Packaging of the helper phage is less efficient than that of pMS, resulting in a mixed phage population that contains predominately recombinant pMS phages.
  • the ends of the phage pick up minor coat proteins specific to each end.
  • the gene III product which is present in three to five copies at one end of the phage.
  • the gene III product is 406 amino acid residues and is required for phage infection of E. coli via the F pili.
  • the first two domains of the heavy chain, the variable and the CH1 domain, are fused to the carboxy- terminal half of the gene III protein.
  • This recombinant pili protein, directed by the pel B leader, is secreted to the peroplasm where it accumulates and forms disulfide bonds with the light chain before it is incorporated in the coat of the phage.
  • another vector contains a FLAG sequence engineered downstream of the gene III.
  • the FLAG is an 8 amino acid peptide expressed at the carboxy terminal of the Fd protein.
  • PCR parameters were optimized to obtain strong enough signals from each primer pair so that ample material was available for cloning of the library.
  • Bone marrow biopsies were taken from CD4 immune monkeys as the source of immunoglobulin RNA. The libraries contained approximately 10 6 members and are currently being panned for specific binders on antigen coated wells.
  • the B7.1 fusion protein was generated similarly, except that the PCR amplified B7.1 gene was cloned into a NEOSPLA cassette vector containing the human CH2 and CH3 immunoglobulin genes. CHO cells were transformed with the B7.1/lg NEOSPLA DNA and stable clones secreting B7.1/lg fusion protein were amplified.
  • the B7.2 and CTLA4 reagents were generated in the same manner, except that for B7.2 the RNA was isolated from human spleen cells that had been stimulated 24 hours with anti-lg and IL-4, and for the CTLA4 constructs the gene source was PHA activated human T cells.
  • Data are mean values of duplicate assays and represent cpm SB7-1 125 bound.
  • the murine MAb L307.4 was inhibitory at concentrations of 10 ⁇ g/ml. Other monkey sera tested at these concentrations were negative (data not shown). These results demonstrate that at least three of the monkeys immunized with both soluble and membrane associated forms of the B7 antigen are producing B7-blocking antibodies with immunosuppressive potential.
  • Antibodies raised against B7.1 are to be tested for cross-reactivity to B7.2. Preliminary results using B7.1 affinity-purified antibodies from B7.1 immune sera provided suggestive evidence of binding to B7.2 transfected CHO cells (not shown). These data should be confirmed by using soluble B7.2lg reagents. We will first purify additional monkey antibodies from B7.1 immunized animals by affinity chromatography on B7.1 Ig-sepharose. We will then produce and purify B7.2lg from CHO cells in sufficient quantities to prepare a B7.2lg-sepharose affinity column. We will select from the B7.1 specific antibody population those antibodies which cross-react with B7.2 by binding to the B7.2lg-sepharose column. Any cross-reactive antibodies identified will be further characterized by direct binding to both B7.1 and B7.2 transfected CHO cells and inhibition of binding to B7.2 transfected cells by B7.1lg.
  • RNA is isolated from the lymphocytes using the method described by Chomczynski Anal. Biochem.. 162(1), 156-159, (1987). RNA is converted to cDNA using an oligo dT primer and reverse transcriptase.
  • the first strand cDNA is divided into aliquots and PCR amplified using the sets of kappa, lambda, and heavy chain Fd region primers described earlier and either Pfu polymerase (Stratagene, San Diego) or Taq polymerase (Promega, Madison).
  • the heavy chain PCR amplified products are pooled, cut with Xho VSpe I restriction enzymes and cloned into the vector pMS. Subsequently, the light chain PCR products are pooled, cut with Sac l/Xba I restriction enzymes, and cloned to create the recombinant library.
  • XLI-Blue E E.
  • coli is transformed with the library DNA and super-infected with VCSM13 to produce the phage displaying antibodies.
  • the library is panned four rounds on polystyrene wells coated with B7.1 or B7.2 antigen. Individual phage clones from each round of panning are analyzed.
  • the pMS vector DNA is isolated and the gene III excised. Soluble Fab fragments are generated and tested in ELISA for binding to B7.1 and B7.2.
  • the monkey phage Fab fragments are characterized for their specificity and the ability to block B7.1-lg and B7.2-lg binding to CTLA-4-lg or CTLA-4 transfected cells. Phage fragments are also characterized for cross-reactivity after first panning for 4 rounds on the B7 species used for immunization in order to select for high affinity fragments. Fab fragments identified from four rounds of panning either on B7.1 or B7.2 antigen coated surfaces are scaled up by infection and grown in 24 hour fermentation cultures of E coli. Fragments are purified by Kodak FLAG binding to a anti-FLAG affinity column.
  • phage Fabs are tested for affinity by an ELISA based direct binding modified Scatchard analysis (Katoh et al, J. Chem. BioEn ⁇ ., 76:451-454, (1993)) using Goat anti-monkey Fab antibodies or anti-FLAG MAb conjugated with horseradish peroxidase.
  • the anti- monkey Fab reagents will be absorbed against human heavy chain constant region Ig to remove any cross-reactivity to B7-lg. Kd values are calculated for each fragment after measurements of direct binding to B7.1-lg or B7.2-lg coated plates.
  • Fab fragments most effectively blocking the binding of B7-lg at the lowest concentrations are selected as lead candidates. Selections are made by competing off 125 l-B7-lg binding to CTLA-4-lg or CTLA-4 transfected cells. Additional selection criteria include, blocking of mixed lymphocyte reaction (MLR), as measured by inhibiting 3H-thymidine uptake in responder cells (Azuma et al, Exp. Med.. 177:845-850,; Azuma et al, Nature, 301:76-79, (1993)) and direct analysis of IL-2 production using IL-2 assay kits. The three or four candidates which are most effective in inhibiting of MLR and CTLA-4 binding assays are chosen for cloning into the above-described mammalian expression vector for transfection into CHO cells and expression of chimeric monkey/human antibodies.
  • MLR mixed lymphocyte reaction
  • Monkey heterohybridomas secreting monoclonal antibodies are generated from existing immunized animals whose sera tested positive for B7.1 and/or B7.2. Lymph node biopsies are taken from animals positive to either, or both, antigens. The method of hybridoma production is similar to the established method used for the generation of monkey anti-CD4 antibodies (Newman, 1992(ld.)). Monkeys with high serum titers will have sections of inguinal lymph nodes removed under anesthesia. Lymphocytes are washed from the tissue and fused with KH6/B5 heteromyeloma cells (Carrol et al, J. Immunol. Meth., 89:61-72, (1986)) using polyethylene glycol (PEG). Hybridomas are selected on H.A.T. media and stabilized by repeated subcloning in 96 well plates.
  • Monkey monoclonal antibodies specific for B7.1 antigen are screened for cross-reactivity to B7.2.
  • Monkey anti-B7 antibodies will be characterized for blocking of B7/CTLA-4 binding using the 125 l-B7-lg binding assay. Inhibition of MLR by 3H-Thymidine uptake and direct measurement of IL-2 production is used to select three candidates. Two candidates will be brought forward in Phase II studies and expressed in CHO cells while repeating all functional studies.
  • anti-B7 antibodies will be tested on cells of several animal species. The establishment of an animal model will allow preclinical studies to be carried out for the selected clinical indication.
  • the antibodies were tested in vitro in a mixed lymphocyte reaction assay (MLR).
  • MLR mixed lymphocyte reaction assay
  • the monkey anti-B7.1 antibodies were tested for their ability to bind B7 on human peripheral blood lymphocytes (PBL). FACS analysis showed that all 4 monkey antibodies tested positive. Monkey antibodies 16C10, 7B6, 7C10 and 20C9 were tested for C1q binding by FACS analysis. Results showed 7C10 monkey Ig had strong human C1q binding after incubating with B7.1 CHO-transfected cells. 16C10 was negative, as were the 20C9 and 7B6 monkey antibodies.
  • CD20- and B7-expressing B-lymphoma cell lines were cultured in complete medium.
  • Complete medium is RPM1 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat inactivated FBS (Hyclone), 2 mM l-glutamine, 100 units/ml of penicillin, and 100 ug/ml of streptomycin.
  • the SKW cell line is Epstein-Barr virus (EBV) positive and can be induced to secrete IgM (SKW 6.4, ATCC).
  • the SB cell line originated from a patient with acute lymphoblastic leukemia and is positive for EBV (CCL-120, ATCC).
  • the Daudi cell line was isolated from a patient with Burkitt's lymphoma (CCL-213, ATCC).
  • Neomycin resistant CD80-expressing Chinese hamster ovary cells were generated using IDEC Pharmaceuticals proprietary vector system.
  • IDEC-114 is a PRIMATIZED ® anti-human CD80 mAb that contains human gamma 1 heavy chain (Lot 114S004F, code 3002G710; Lot ZPPB-01) and rituximab is an anti-human CD20 specific mouse-human gamma 1 chimeric antibody (Lot E9107A1 ; Lot D9097A1).
  • antibodies used include the murine anti-human CD80 mAb L307.4 (BD Pharmingen, San Diego, CA), the primatized anti-human CD4 mAb CE9.1 , with human gamma 1 chain (Lot M2CD4156), and the murine isotype-matched (lgG1) control antibody 3C9 developed at IDEC Pharmaceuticals.
  • Binding of antibodies to CD20 and CD80 molecules expressed on different B-lymphoma lines was determined by flow cytometry. Varying concentrations of test or control antibodies diluted in cold fluorescence-activated cell sorting (FACS) binding buffer was incubated in a cell-culture tube with 1 x 10 6 cells to a final volume of 200 ⁇ l. IDEC-114 and rituximab were used as test antibodies and CE9.1 was used as the isotype-matched negative control. The cells were incubated for 60 minutes on ice and washed once in FACS wash buffer following incubation.
  • FACS cold fluorescence-activated cell sorting
  • PBMC peripheral monocytes
  • Isotype matched CE9.1 (Lot M2CD4156) or L307.4 (BD Pharmingen), or a murine isotype-matched (IgG-i) antibody of irrelevant specificity, 3C9, were used. All wells were plated in triplicate into a 96 well, round bottom tissue culture plate. The effector cells were harvested, washed once with complete medium, and added at 1 x 10 6 cells in 100 ⁇ l volume per well to obtain a 50:1 effector to target ratio. The following control wells were also included in triplicate: target cell incubated with 100 ⁇ l complete medium to determine spontaneous release and target cell incubated with 100 ⁇ l 0.5% Triton X-100 (Sigma-Aldrich Corp.) to determine maximum release.
  • the culture was incubated for 4 hours at 37°C and 5% CO 2 and the 51 Cr released in the culture supernatant due to cell lysis was determined by a gamma counter (ISODATA).
  • the cytotoxicity was expressed as the percentage of specific lysis and calculated as follows: 51 Cr release of test samples - spontaneous
  • the CDC activity of IDEC-114 and rituximab was determined using B-cell lines and human complement (C). Dilutions of antibodies were made at 4x concentration and 50 ⁇ l was dispensed into each 96 well in triplicates. The SKW or Daudi cells were labeled with 51 Cr (150 ⁇ Ci/10 6 cells) for 1 hour at 37°C and 5% CO 2 . The cells were washed four times and resuspended in complete medium, and 1 x 10 4 cells in 50 ⁇ l were dispensed into each well. One hundred ⁇ l of normal human serum complement (Quidel, San Diego, CA) diluted 1 :4 or 1:8 in complete medium was added.
  • a human lymphoma tumor model in severe immunodeficiency (SCID) mice was developed. Briefly, 3 x 10 6 to 4 x 10 6 SKW cells were intravenously injected into 6- to 8-week old female SCID mice and their survival was monitored for 45 to 60 days. All mice developed a paralytic form of the disease before circumventing to death. Mice that developed severe paralysis were sacrificed and scored as dead. Kaplan-Meier analysis was performed using the Statistical Analysis System (SAS) and p-values were generated by the Log-rank test.
  • SAS Statistical Analysis System
  • CD80 is transiently expressed on the surface of activated B cells and activated APCs, but is weakly expressed or not expressed on resting B-cells and resting APCs. Since CD80 is a B-cell activation marker, it is expressed primarily on the dividing and/or activated lymphoma cells. Reports suggest that CD80 is consfitutively expressed on malignant B cells. To confirm these reports, we tested the expression of CD80 by flow cytometry in a panel of lymphoma and leukemia specimens obtained from 20 patients. Results indicate that CD80 is expressed in lymphomas and leukemias at different densities (Table 3).
  • CD80 expression on follicular, small-cleaved, low-grade lymphoma samples is presented in Table 4.
  • the CD80 expression on these lymphoma cells ranged from 25% to 90% of the tumor cells in the samples. It is interesting, however, that within the same lymphoma the "large” cells were 90% to 100% positive, while the "small” cells were 25% to 100% positive. It is possible that CD80 is expressed on proliferating or activated malignant B cells, which may account for the variability of expression within lymphoma samples tested.
  • IDEC-114 The binding activity of IDEC-114 to CD80 expressed on CD80-CHO transfectant and on lymphoma cell lines was determined by flow cytometry shows the specific binding of IDEC-114 from two different lots (Lot 114S004F and Lot 114S015) to CD80-CHO cells in a concentration dependent fashion. As expected, isotype-matched control antibody of irrelevant specificity (IDEC-152) did not bind to CD80-CHO cells. Testing of IDEC-114 for binding to CD80 on SKW and SB lymphoma cell lines showed a lower binding than that of rituximab as demonstrated by a lower percentage of positive cells (Table 5) and lower mean fluorescence intensity (Table 6).
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • FIG. 11 shows the ADCC activity of IDEC-114 and rituximab on CD20 + /CD80 + SB and SKW cells. Overall, higher levels of ADCC activity were observed with SB cells than with SKW cells. IDEC-114 showed a dose-dependent killing of SB and SKW cells with a maximum killing of 75% and 46%, respectively, at 10 ⁇ g/ml.
  • Rituximab at comparable antibody concentrations showed higher ADCC activity (97% on SB cells and 65% on SKW cells) than IDEC-114, which correlated with higher cell-binding activity of rituximab compared with IDEC-114.
  • murine L307.4 which does not bind to the human Fc receptor, showed weak ADCC activity. Only background levels of ADCC were observed with isotype human and murine controls (CE9.1 and 3C9, respectively).
  • lymphoma cells have been reported to be resistant to complement-mediated killing. Resistance was associated with the cell-surface expression of C-regulatory proteins such as CD55 and CD59 antigens. Flow cytometric analysis demonstrated higher expression of CD55 on SKW cells compared with Daudi cells (results not shown), which correlated with lower cytolytic activity of rituximab on SKW cells.
  • IDEC-114 Based on the antitumor activity of IDEC-114 as a single agent, a combination of IDEC-114 and rituximab was evaluated in the same tumor model at the same dosing schedule described in previously. (In Vivo Therapeutic Effect of IDEC-114 and Rituximab Single-Agent Therapies in Lymphoma). SKW/SCID mice were injected with 200 ⁇ g of IDEC-114 and 200 ⁇ g of rituximab, and compared with the mice injected with either 200 ⁇ g or 400 ⁇ g of IDEC-114 or 200 ⁇ g or 400 ⁇ g of rituximab.
  • Figure 16 shows the survival advantage of mice treated with a IDEC-114/rituximab combination therapy compared with mice treated with either IDEC-114 or rituximab as a single-agent therapy. Results show that the combination of IDEC-114 and rituximab leads to increased disease-free survival compared with either antibody alone. In the combination therapy group, 70% (7/10) of the mice survived for more than 50 days after the last antibody injection. In contrast, less than 10% of mice treated with IDEC-114 or rituximab alone were alive at the end of the study. Survival data were analyzed by Kaplan-Meier and Log-rank tests (Table 7).
  • the IDEC-114/rituximab combination therapy produced a statistically greater response than 200 ⁇ g or 400 ⁇ g of IDEC-114 or rituximab single-agent therapy.
  • Table 7 Comparison of IDEC-114/Rituximab Combination Therapy with IDEC-114 or
  • Combination treatment of IDEC-114 with adriamycin provide increased disease-free survival in human lymphoma-muse model
  • IDEC-114 as single-agent therapy showed delay in disease progression with 50% survival at day 38, whereas, ADM at 2.5mg/Kg dose was effective with 50% survival at day 41. No anti-tumor effect was observed with ADM at 1.25 mg/Kg dose. The anti-tumor response observed with IDEC-114 was comparable to the anti-tumor response of ADM at 2.5mg/Kg.
  • a combination of IDEC-114 and ADM was evaluated in the same tumor model at the same dosing schedule. Mice were injected with combination of 100 ⁇ g of IDEC-114 and ADM at 1.25mg/Kg or 2.5mg/Kg doses.
  • Results show that the combination of IDEC-114 and with 2.5mg/Kg leads to increased disease-free survival compared with either IDEC-114 or 2.5mg/Kg ADM alone.
  • 77% (7/9) of the mice survived at the end of the study.
  • less than 25% of mice treated with IDEC-114 or ADM alone were alive at the end of the study.
  • Combination of 100ug dose of IDEC-114 with 1.25 mg/Kg ADM did not show increased in survival.
  • the IDEC-114/ADM (2.5mg/kg) combination therapy produced a statistically greater response than 100ug of IDEC-114 or 2.5 mg/Kg ADM single-agent therapy.
  • CD80 can be used as a target for antibody therapy of lymphoma.
  • antibodies against CD80 have been shown to synergize with rituximab by increasing the antibody density on the lymphoma cells rendering them more susceptible to killing mechanisms such as ADCC.
  • IDEC-114 in a disseminated human B lymphoma/SCID mouse model, IDEC-114 as a single-agent therapy showed antitumor activity comparable to that of rituximab.

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Abstract

Procédés pour traiter des tumeurs malignes comportant des cellules B, notamment la leucémie et le lymphome à cellules B, au moyen d'un anticorps anti-CD80 isolément ou en combinaison avec un anticorps anti-CD80 et la chimiothérapie. Ces procédés permettent une réponse antitumorale synergique.
PCT/US2002/036226 2001-11-09 2002-11-12 Anticorps anti-cd80 presentant une activite adcc visant la mort des cellules b de lymphome a mediation par adcc, isolement ou en combinaison avec d'autres therapies WO2003039486A2 (fr)

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WO2006029224A3 (fr) * 2004-09-08 2006-09-08 Genentech Inc Procedes d'utilisation de ligands des recepteurs de mort et d'anticorps cd20
EP2123676A4 (fr) * 2007-01-05 2011-01-05 Univ Tokyo Diagnostic et traitement de cancers utilisant un anticorps anti-prg-3
WO2012121775A2 (fr) 2010-12-21 2012-09-13 Abbott Laboratories Immunoglobulines à double domaine variable et leurs utilisations
WO2013063095A1 (fr) 2011-10-24 2013-05-02 Abbvie Inc. Agents de liaison immunologique dirigés contre la sclérostine
US8586714B2 (en) 2009-09-01 2013-11-19 Abbvie, Inc. Dual variable domain immunoglobulins and uses thereof
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US9029508B2 (en) 2008-04-29 2015-05-12 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
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US9109026B2 (en) 2008-06-03 2015-08-18 Abbvie, Inc. Dual variable domain immunoglobulins and uses thereof
US9120870B2 (en) 2011-12-30 2015-09-01 Abbvie Inc. Dual specific binding proteins directed against IL-13 and IL-17
EP2921177A2 (fr) 2010-07-09 2015-09-23 AbbVie Inc. Immunoglobulines à double domaine variable et utilisations de celles-ci
US9303085B2 (en) 2010-05-14 2016-04-05 Abbvie Inc. IL-1 binding proteins
EP2483304B1 (fr) 2009-09-29 2016-05-04 F.Hoffmann-La Roche Ag Réglage de filtration préalable de solutés issus de tampon pour la preparation a forte teneur en immunoglobuline
US9670276B2 (en) 2012-07-12 2017-06-06 Abbvie Inc. IL-1 binding proteins
US9840554B2 (en) 2015-06-15 2017-12-12 Abbvie Inc. Antibodies against platelet-derived growth factor (PDGF)
WO2017218698A1 (fr) 2016-06-15 2017-12-21 Sutro Biopharma, Inc. Anticorps à domaines ch2 modifiés, compositions les contenant et leurs procédés d'utilisation
US10093733B2 (en) 2014-12-11 2018-10-09 Abbvie Inc. LRP-8 binding dual variable domain immunoglobulin proteins
US10273281B2 (en) 2015-11-02 2019-04-30 Five Prime Therapeutics, Inc. CD80 extracellular domain polypeptides and their use in cancer treatment
WO2021178597A1 (fr) 2020-03-03 2021-09-10 Sutro Biopharma, Inc. Anticorps comprenant des étiquettes de glutamine spécifiques à un site, leurs procédés de préparation et d'utilisation
US11789010B2 (en) 2017-04-28 2023-10-17 Five Prime Therapeutics, Inc. Methods of treatment with CD80 extracellular domain polypeptides

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CA2390412A1 (fr) * 1999-11-08 2001-05-17 Idec Pharmaceuticals Corporation Traitement de tumeurs malignes des cellules b a l'aide d'anticorps anti-cd40l associes a des anticorps anti-cd20, et/ou chimiotherapie et radiotherapie
WO2001074388A1 (fr) * 2000-03-31 2001-10-11 Idec Pharmaceuticals Corporation Utilisation combinee d'anticorps ou d'antagonistes anti-cytokine et d'anti-cd20 pour le traitement du lymphome b

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EP2123676A4 (fr) * 2007-01-05 2011-01-05 Univ Tokyo Diagnostic et traitement de cancers utilisant un anticorps anti-prg-3
EP2899209A1 (fr) 2008-04-29 2015-07-29 Abbvie Inc. Immunoglobuline à double domaine variable et ses utilisations
US9029508B2 (en) 2008-04-29 2015-05-12 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
EP3002299A1 (fr) 2008-06-03 2016-04-06 AbbVie Inc. Immunoglobulines à deux domaines variables et leurs utilisations
US9109026B2 (en) 2008-06-03 2015-08-18 Abbvie, Inc. Dual variable domain immunoglobulins and uses thereof
US9035027B2 (en) 2008-06-03 2015-05-19 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
US8822645B2 (en) 2008-07-08 2014-09-02 Abbvie Inc. Prostaglandin E2 dual variable domain immunoglobulins and uses thereof
EP2772269A2 (fr) 2009-03-05 2014-09-03 Abbvie Inc. Protéines se liant à un IL-17
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US9663587B2 (en) 2009-03-05 2017-05-30 Abbvie Inc. IL-17 binding proteins
US8586714B2 (en) 2009-09-01 2013-11-19 Abbvie, Inc. Dual variable domain immunoglobulins and uses thereof
EP2483304B1 (fr) 2009-09-29 2016-05-04 F.Hoffmann-La Roche Ag Réglage de filtration préalable de solutés issus de tampon pour la preparation a forte teneur en immunoglobuline
US8716450B2 (en) 2009-10-15 2014-05-06 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
US8722855B2 (en) 2009-10-28 2014-05-13 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
US9303085B2 (en) 2010-05-14 2016-04-05 Abbvie Inc. IL-1 binding proteins
EP2921177A2 (fr) 2010-07-09 2015-09-23 AbbVie Inc. Immunoglobulines à double domaine variable et utilisations de celles-ci
EP3252072A2 (fr) 2010-08-03 2017-12-06 AbbVie Inc. Immunoglobuline à double domaine variable et ses utilisations
US9493560B2 (en) 2010-08-03 2016-11-15 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
US8735546B2 (en) 2010-08-03 2014-05-27 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
US9046513B2 (en) 2010-08-26 2015-06-02 Abbvie Inc. Dual variable domain immunoglobulins and uses thereof
WO2012121775A2 (fr) 2010-12-21 2012-09-13 Abbott Laboratories Immunoglobulines à double domaine variable et leurs utilisations
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US9120870B2 (en) 2011-12-30 2015-09-01 Abbvie Inc. Dual specific binding proteins directed against IL-13 and IL-17
WO2013185115A1 (fr) 2012-06-08 2013-12-12 Sutro Biopharma, Inc. Anticorps comprenant des résidus d'acides aminés non endogènes spécifiques d'un site, leurs procédés de préparation et leurs procédés d'utilisation
EP3505534A1 (fr) 2012-06-08 2019-07-03 Sutro Biopharma, Inc. Anticorps comprenant des résidus d'acides aminés non endogènes spécifiques d'un site, leurs procédés de préparation et leurs procédés d'utilisation
US9670276B2 (en) 2012-07-12 2017-06-06 Abbvie Inc. IL-1 binding proteins
WO2014036492A1 (fr) 2012-08-31 2014-03-06 Sutro Biopharma, Inc. Acides aminés modifiés comprenant un groupe azido
EP3584255A1 (fr) 2012-08-31 2019-12-25 Sutro Biopharma, Inc. Acides aminés modifiés comprenant un groupe azido
EP4074728A1 (fr) 2012-08-31 2022-10-19 Sutro Biopharma, Inc. Peptides modifiés comprenant un groupe azido
US9045551B2 (en) 2012-11-01 2015-06-02 Abbvie Inc. Anti-DLL4/VEGF dual variable domain immunoglobulin and uses thereof
US9944720B2 (en) 2012-11-01 2018-04-17 Abbvie Inc. Anti-DLL4/VEGF dual variable domain immunoglobulin and uses thereof
US9163093B2 (en) 2012-11-01 2015-10-20 Abbvie Inc. Anti-DLL4/VEGF dual variable domain immunoglobulin and uses thereof
US9062108B2 (en) 2013-03-15 2015-06-23 Abbvie Inc. Dual specific binding proteins directed against IL-1 and/or IL-17
US8987418B2 (en) 2013-03-15 2015-03-24 Abbvie Inc. Dual specific binding proteins directed against IL-1β and/or IL-17
WO2015006555A2 (fr) 2013-07-10 2015-01-15 Sutro Biopharma, Inc. Anticorps comprenant plusieurs résidus d'acides aminés non naturels site-spécifiques, des procédés permettant leur préparation et leurs méthodes d'utilisation
EP3336103A1 (fr) 2013-07-10 2018-06-20 Sutro Biopharma, Inc. Anticorps comprenant plusieurs résidus d'acides aminés non naturels sitespécifiques, des procédés permettant leur préparation et leurs méthodes d'utilisation
WO2015054658A1 (fr) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Acides aminés modifiés comprenant des groupes fonctionnels de tétrazine, procédés de préparation et procédés d'utilisation associés
US10093733B2 (en) 2014-12-11 2018-10-09 Abbvie Inc. LRP-8 binding dual variable domain immunoglobulin proteins
US9840554B2 (en) 2015-06-15 2017-12-12 Abbvie Inc. Antibodies against platelet-derived growth factor (PDGF)
US10273281B2 (en) 2015-11-02 2019-04-30 Five Prime Therapeutics, Inc. CD80 extracellular domain polypeptides and their use in cancer treatment
US11098103B2 (en) 2015-11-02 2021-08-24 Five Prime Therapeutics, Inc. CD80 extracellular domain polypeptides and their use in cancer treatment
WO2017218698A1 (fr) 2016-06-15 2017-12-21 Sutro Biopharma, Inc. Anticorps à domaines ch2 modifiés, compositions les contenant et leurs procédés d'utilisation
US11789010B2 (en) 2017-04-28 2023-10-17 Five Prime Therapeutics, Inc. Methods of treatment with CD80 extracellular domain polypeptides
WO2021178597A1 (fr) 2020-03-03 2021-09-10 Sutro Biopharma, Inc. Anticorps comprenant des étiquettes de glutamine spécifiques à un site, leurs procédés de préparation et d'utilisation

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