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WO2001089567A1 - Identification d'interactions de liaison unique entre certains anticorps et les antigenes humains co-stimulateurs b7.1 et b7.2 - Google Patents

Identification d'interactions de liaison unique entre certains anticorps et les antigenes humains co-stimulateurs b7.1 et b7.2 Download PDF

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WO2001089567A1
WO2001089567A1 PCT/US2001/016364 US0116364W WO0189567A1 WO 2001089567 A1 WO2001089567 A1 WO 2001089567A1 US 0116364 W US0116364 W US 0116364W WO 0189567 A1 WO0189567 A1 WO 0189567A1
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cells
antibody
antigen
human
antibodies
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Darrell R. Anderson
Nabil Hanna
Peter Brams
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Idec Pharmaceuticals Corp
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Idec Pharmaceuticals Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • 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'

Definitions

  • the present invention relates to the identification and use of monoclonal antibodies which are specific to B7.1 (CD80) and/or B7.2 (CD86) antigen. More specifically, the present invention relates to the identification and use of monoclonal antibodies or primatized forms thereof which are capable of inhibiting the binding of human B7.1 (CD80) or B7.2 (CD86) antigen to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Thus, the invention relates to the identification and use of monoclonal antibodies and primatized forms thereof which recognize specific sites on the B7.1 (CD80) or B7.2 (CD86) antigen which are not involved in CTLA-4 receptor binding. The invention further relates to monoclonal antibodies or primatized forms thereof which recognize specific sites on the human B7.1 (CD80) and/or B7.2 (CD86) antigen and are capable of inhibiting IL-2 production.
  • the present invention relates to pharmaceutical compositions containing monoclonal or primatized antibodies specific to human B7.1 (CD80) and B7.2 (CD86) and their use as immunosuppressants by modulating the B7:CD28 pathway, e.g., for the treatment of autoimmune disorders, and the prevention of organ rejection.
  • B7:CD28 has been studied by different research groups because of its significant role in B- and T-cell activation.
  • B- and T-cell activation June CH, Bluestone JA, Linsley PS, Thompson CD: "Role of the CD28 receptor in T-cell activation.” Immunol Today 15:321 (1994); June CH, Ledbetter JA: "The role of the CD28 receptor during T-cell responses to antigen.”
  • the elaboration of a successful immune response depends on a series of specific interactions between a T cell and an antigen presenting cell. Although the essential first step in this process depends upon the binding of antigen to the T cell receptor, in the context of the MHC class JJ molecule (Lane, P.J.L., F.M. McConnell, G.L. Schieven, E.A. Clark, and J.A. Ledbetter, (1990), "The Role of Class JJ Molecules in Human B Cell Activation.” The Journal of Immunology 144:3684- 3692), this interaction alone is not sufficient to induce all the events necessary for a sustained response to a given antigen (Schwartz, R.H.
  • B7.1 (CD80) and B7.2 (CD86) expressed on antigen presenting cells are major pairs of costimulatory molecules necessary for a sustained immune response (Azuma, M., H. Yssel, J.H. Phillips, H. Spits, and L.L. Lanier (1993), "Functional Expression of B7/BB1 on Activated T Lymphocytes.” J. Exp. Med. 177:845-850; Freeman, G.J., A.S. Freedman, J.M. Segil, G. Lee, J.F. Whitman, and LM.
  • B7/BB-1 is a Leucocyte Differentiation Antigen on Human Dendritic Cells Induced by Activation.” Immunology 79:616-620). It can be shown in vitro that the absence of these co-stimulatory signals leads to an aborted T cell activation pathway and the development of unresponsiveness to the specific antigen, or anergy. (See, e.g., Harding, F.A., J.G. McArthur, J.A. Gross, D.M. Raulet, and J.P. Allison (1992),
  • the molecules B7.1 (CD80) and B7.2 (CD86) can bind to either CD28 or
  • CTLA-4 although B7.1 (CD80) binds to CD28 with a Kd of 200 Nm and to CTLA-4 with a 20-fold higher affinity (Linsley, P.S., E.A. Clark, and J.A. Ledbetter (1990), "T-Cell Antigen CD28 Mediates Adhesion with B Cells by Interacting with Activation Antigen B7/BB-1.” Proc. Natl. Acad. Sci. 87:5031-5035; Linsley et al (1993), "The Role of the CD28 receptor during T cell responses to antigen," Annu. Rev. Immunol.
  • B7.1 is expressed on activated B cells and interferon induced monocytes, but not resting B cells (Freeman, G.J., G.S. Gray, CD. Gimmi, D.B. Lomarrd, L-J. Zhou, M. White, J.D. Fingeroth, J.G. Gribben, and LM. Nadler (1991).
  • B7.2 (CD86), on the other hand, is constitutively expressed at very low levels on resting monocytes, dendritic cells and B cells, and its expression is enhanced on activated T cells, NK cells and B lymphocytes (Azuma, M. D. Ito, H. Yagita, K. Okumura, J.H. Phillips, L.L. Lanier, and C. Somoza 1993, "B70 Antigen is a Second Ligand for CTLA-4 and CD28," Nature, 366:76-79).
  • B7.1 and B7.2 can be expressed on the same cell type, their expression on B cells occurs with different kinetics (Lenschow, D.J., G.H. Su, L.A. Zuckerman, N. Nabavi, C.L. Jellis, G.S. Gray, J. Miller, and J.A. Bluestone (1993), "Expression and Functional Significance of an Additional Ligand for CTLA-4," Proc. Natl. Acad. Sci., USA, 90:11054-11058; Boussiotis, V.A., G.J. Freeman, J.G. Gribben, J. Daley, G. Gray, and L.M.
  • CTLA-4/CD28 counter receptors may be expressed at various times after B Cell activation. More recently, it has been suggested that the second T cell associated co- receptor CTLA-4 apparently functions as a negative modulator to override and prevent a runaway immune system (Krummel M, Allison J: “CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation.” J. Exp. Med. 182:459-466 (1995)).
  • CTLA-4 receptor plays a critical role in down regulating the immune response, as evidenced in CTLA-4 knockout mice.
  • Knockout mice born without the ability to express the CTLA-4 gene die within 3-4 weeks of severe lymphoproliferative disorder (Tivol EA, Borriello G, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH: "Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4.” Immunity 3:541-547 (1995)).
  • CTLA-4 is thought to function through signaling mechanisms linked to induction of apoptosis (Gribben JG, Freeman GJ, Boussiotis VA, Rennert P, Jellis CL, Greenfield E, Barber M, Restivo Jr. VA, Ke X, Gray GS, Nadler LM: "CTLA-4 mediates antigen specific apoptosis of human T cells.” Proc. Natl. Acad. Sci. USA 92:811-815 (1995)), triggered through as yet undefined ligand binding to specific cites on the receptor.
  • both B7.1 (CD80) and B7.2 (CD86) contain extracellular immunoglobulin superfamily V and C-like domains, a hydrophobic transmembrane region and a cytoplasmic tail (Freeman, G.J., J.G. Gribben, V.A. Boussiotis, J.W. Ng, V. Restivo, Jr., L.A. Lombard, G.S. Gray, and L.M. Nadler (1993), "Cloning of B7.2: A CTLA-4 Counter-receptor that Co-stimulates Human T Cell Proliferation," Science 262:909). Both B7.1 and B7.2 are heavily glycosylated.
  • B7.1 is a 44-54kD glycoprotein comprised of a 223 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 61 amino acid cytoplasmic tail.
  • B7.1 (CD80) contains 3 potential protein kinase phosphorylation sites. (Azuma, M., H. Yssel, J.H. Phillips, H. Spits, and L.L. Lanier, (1993), "Functional Expression of B7/BB1 on Activated T Lymphocytes," J. Exp. Med. 177:845-850).
  • B7.2 (CD86) is a 306 amino acid membrane glycoprotein.
  • Blockade of the B7/CD28 Interaction Blocking of the B7/CD28 (CD80 and/or CD86) interaction offers the possibility of inducing specific immunosuppression, with potential for generating long lasting antigen-specific therapeutic effects. Antibodies or agents that temporarily prevent this interaction may be useful, specific and safe clinical immunosuppressive agents, with potential for generating long term antigen-specific therapeutic effects. Antibodies to either B7.1 (CD80) or B7.2 (CD86) have been shown to block T cell activation, as measured by the inhibition of IL-2 production in vitro (DeBoer, M., P. Parren, J. Dove, F. Ossendorp, G. van der Horst, and J.
  • CTLA-4Ig fusion protein and anti-CD28 Fabs were shown to have similar effects on the down regulation of JL-2 production. In vivo administration of a soluble CTLA-4Ig fusion protein has been shown to suppress T cell dependent antibody responses in mice (Linsley, P.S., J.L. Greene, P. Tan, J.
  • CTLA-4Ig was able to prevent pancreatic islet cell rejection in mice by directly inhibiting the interaction of T cells and B7.1/B7.2 antigen presenting cells (Lenschow, D.J., G.H. Su, L.A. Zuckerman, N. Nabavi, C.L. Jellis, G.S. Gray, J. Miller, and J.A. Bluestone (1993), "Expression and Functional Significance of an Additional Ligand for CTLA-4," Proc. Natl. Acad. Sci., USA 90:11054-11058). In this case, long term donor specific tolerance was achieved. 3. Recombinant Phage Display Technology for Antibody Selection
  • Phage display technology is beginning to replace traditional methods for isolating antibodies generated during the immune response, because a much greater percentage of the immune repertoire can be assessed than is possible using traditional methods. This is in part due to PEG fusion inefficiency, chromosomal instability, and the large amount of tissue culture and screening associated with heterohybridoma production. Phage display technology, by contrast, relies on molecular techniques for potentially capturing the entire repertoire of immunoglobulin genes associated with the response to a given antigen. This technique is described by Barbas et al, Proc. Natl. Acad. Sci, USA 88:7978-7982 (1991).
  • immunoglobulin heavy chain genes are PCR amplified and cloned into a vector containing the gene encoding the minor coat protein of the filamentous phage M13 in such a way that a heavy chain fusion protein is created.
  • the heavy chain fusion protein is incorporated into the Ml 3 phage particle together with the light chain genes as it assembles.
  • Each recombinant phage contains, within its genome, the genes for a different antibody Fab molecule which it displays on its surface. Within these libraries, in excess of 10 different antibodies can be cloned and displayed.
  • the phage library is panned on antigen coated microliter wells, non-specific phage are washed off, and antigen binding phage are eluted.
  • This technique modifies antibodies such that they are not antigenically rejected upon administration in humans.
  • This technique relies on immunization of cynomolgus monkeys with human antigens or receptors. This technique was developed to create high affinity monoclonal antibodies directed to human cell surface antigens.
  • the technology relies on the fact that despite the fact that cynomolgus monkeys are phylogenetically similar to humans, they still recognize many human proteins as foreign and therefore mount an immune response. Moreover, because the cynomolgus monkeys are phylogenetically close to humans, the antibodies generated in these monkeys have been discovered to have a high degree of amino acid homology to those produced in humans. Indeed, after sequencing macaque immunoglobulin light and heavy chain variable region genes, it was found that the sequence of each gene family was 85-98% homologous to its human counterpart (Newman et al, (1992), Id.). The first antibody generated in this way, an anti-CD4 antibody, was 91-92% homologous to the consensus sequence of human immunoglobulin framework regions. Newman et al, Biotechnology 10:1458-1460 (1992).
  • Evidence is provided in this disclosure for the identification of monoclonal antibodies which recognize specific sites on the B7.1 antigen which are restricted to CD28 receptor binding. Furthermore, evidence is presented herein for the identification of antibodies which recognize sites on the B7.1 and/or B7.2 antigen which are exclusive of CTLA-4 receptor binding. Thus, evidence is presented herein to support the existence of unique antigen binding sites on the human B7.1 (CD80) co-stimulatory antigen. The sites claimed are identified by anti-B7.1 PRIMATIZED® antibodies and evidence is presented which confirms binding to a site of interaction on the B7.1 antigen which is restricted to binding with the co-activation receptor CD28.
  • An object of the invention is to identify novel antibodies which are specific to human B7.1 antigen. More specifically, it is an object of the invention to identify antibodies which are specific to human B7.1 antigen and which are also capable of inhibiting the binding of B7.1 to a CD28 receptor. It is also an object of this invention to identify antibodies which are specific to human B7.1 antigen and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Thus, an object of this invention is to identify antibodies which recognize specific sites on the B7.1 antigen, wherein the recognized sites are restricted to CD28 receptor binding and which are exclusive of CTLA-4 receptor binding. It is a further object of the invention to identify antibodies which are specific to human B7.1 (CD80) antigen and which fail to recognize human B7.2 (CD86) antigen.
  • immunosuppressants i.e., to block antigen driven immune responses
  • SLE systemic erythematosus
  • type 1 diabetes mellitus idiopathic thrombocytopema purpura
  • ITP idiopathic thrombocytopema pur
  • compositions containing one or more monoclonal antibodies specific to human B7.1 (CD80) antigen or primatized forms thereof, and a pharmaceutically acceptable carrier or excipient.
  • These compositions will be used, e.g., as immunosuppressants to treat autoimmune diseases, e.g., idiopathic thrombocytopema purpura (ITP) and systemic lupus erythematosus (SLE), to block antigen driven immune responses, and to prevent organ rejection in transplant recipients.
  • autoimmune diseases e.g., idiopathic thrombocytopema purpura (ITP) and systemic lupus erythematosus (SLE)
  • ITP idiopathic thrombocytopema purpura
  • SLE systemic lupus erythematosus
  • B7:CD28 pathway e.g., autoimmune diseases such as idiopathic thrombocytopema purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, psoriasis, rheumatoid arthritis, multiple sclerosis, aplastic anemia, as well as for preventing rej ection in transplantation subj ects .
  • ITP idiopathic thrombocytopema purpura
  • SLE systemic lupus erythematosus
  • type 1 diabetes mellitus psoriasis
  • rheumatoid arthritis rheumatoid arthritis
  • multiple sclerosis aplastic anemia
  • transfectants e.g., CHO cells, which express at least the variable heavy and light domains of monoclonal antibodies specific to the human B7.1 antigen and/or B7.2 antigen.
  • Non-depletin antibody - an antibody which blocks the co-stimulatory action of B7 and T cell activating ligands CD28 and CTLA-4. Thus, it anergizes but does not eliminate the antigen presenting cell.
  • Primatized antibody - a recombinant antibody which has been engineered to contain the variable heavy and light domains of a monkey antibody, in particular, a cynomolgus monkey antibody, and which contains human constant domain sequences, preferably the human immunoglobulin gamma 1 or gamma 4 constant domain (or PE variant). The preparation of such antibodies is described in Newman et al, (1992), "Primatization of Recombinant Antibodies for immunotherapy of Human Diseases: A
  • B7 antigens - B7 antigens in this application include, e.g., human B7, B7.1 and B7.2 antigens. These antigens bind to CD28 and/or CTLA-4. These antigens have a co-stimulatory role in T cell activation. Also, these B7 antigens all contain extracellular immunoglobulin superfamily V and C-like domains, a hydrophobic transmembrane region and a cytoplasmic tail (See, Freeman et al, Science 262:909, (1993)), and are heavily glycosylated.
  • Anti-B7 antibodies - Antibodies preferably monkey monoclonal antibodies or primatized forms thereof, which specifically bind human B7 antigens, e.g., human B7.1 and/or B7.2 antigen with a sufficient affinity to block the B7:CD28 interaction, but h do not block the B7/CTLA-4 receptor interaction and thereby induce immunosuppression.
  • human B7 antigens e.g., human B7.1 and/or B7.2 antigen with a sufficient affinity to block the B7:CD28 interaction, but h do not block the B7/CTLA-4 receptor interaction and thereby induce immunosuppression.
  • Apoptosis-inducing Antibody - An antibody that induces programmed cell death (“apoptosis").
  • assays for detecting apoptosis including capsage activation assays.
  • 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 a depicts the amino acid and nucleic acid sequence of a primatized form of the light chain of 7C10.
  • Figure 3b depicts the amino acid and nucleic acid sequence of a primatized form of the heavy chain of 7C 10.
  • Figure 4a depicts the amino acid and nucleic acid sequence of a primatized form of the light chain of 7B6.
  • Figure 4b depicts the amino acid and nucleic acid sequence of a primatized form of the heavy chain of 7B6.
  • Figure 5 a depicts the amino acid and nucleic acid sequence of a primatized light chain 16C10.
  • Figure 5b depicts the amino acid and nucleic acid sequence of a primatized heavy chain 16C10.
  • Figure 6 depicts the inability of PI 6C 10 to block CTLA-4Ig-Biotin binding to B7.1 transfected CHO cells.
  • Figure 7 depicts the inability of CTLA-4Ig to block P16C10-Biotin binding to B7.1 transfected CHO cells.
  • Figure 8 depicts that BB-1 completely blocks binding of CTLA-4Ig-Biotin to B7.1 transfected CHO cells and further depicts the inability of BB-1 to significantly affect P 16C 10-Biotin binding to B7.1 transfected CHO cells.
  • FIG. 9 depicts that CTLA-4Ig-Biotin is effectively blocked by all B7.1 inhibitors except P16C10.
  • Figure 10 depicts the ability of PI 6C 10 to block binding of the CD28/B7-lIg interaction. Data shown are averages of values obtained from four separate experiments.
  • Figure 11 depicts production of IL-2 in cultures of purified normal human CD4+ lymphocytes when stimulated with sub-optimal amounts of immobilized anti- CD3 antibody and B7-1 (CD80) on latex microbeads.
  • L307.4 is a commercially available murine antibody (B/D Pharmingen) that binds specifically to human CD80 and neutralizes CD28:CD80 functional interactions.
  • CTLA-4Ig is a soluble receptor fusion protein that specifically blocks CD80 and CD86 binding to CD28 receptors on T cells.
  • IDEC-114 is a PRIMATIZED monoclonal antibody that specifically binds to both soluble and membrane forms of the CD80 antigen but does not recognize CTLA- 4 or B7-2 antigens.
  • Figure 12 depicts uptake of H3-Thymidine in cultures of purified normal human CD4+ lymphocytes when stimulated with sub-optimal amounts of immobilized anti-CD3 antibody and B7-1 (CD80) on latex microbeads.
  • L307.4 is a commercially available murine antibody (B/D Pharmingen) that binds specifically to human CD80 and neutralizes CD28:CD80 functional interactions.
  • CTLA-4Ig is a soluble receptor fusion protein that specifically blocks CD80 and CD86 binding to CD28 receptors on T cells.
  • IDEC-114 is a PRIMATIZED monoclonal antibody that specifically binds to both soluble and membrane forms of the CD80 antigen but does not recognize CTLA-4 or B7-2 antigens.
  • Figure 13 depicts production of TH2 cytokine IL-10 in cultures of purified normal human CD4+ lymphocytes when stimulated with sub-optimal amounts of immobilized anti-CD3 antibody and B7-1 (CD80) on latex microbeads. Inhibition of IL-10 production by L307.4 anti-CD80 and CTLA-4Ig fusion protein was compared at 0.1,1, and lO ⁇ g/mL.
  • Figure 14 depicts inhibition of IL-2 cytokine production by CTLA-4Ig and
  • IDEC-114 in cultures of purified human CD4+ T cells.
  • T cells were co-stimulated with anti-CD3 and B7Ig coated latex microbeads with an anti-CD3/B7 ratio (w/w) of 8:1.
  • IL-2 was determined by growth and uptake of Thymidine by the IL-2 dependent cell line CTLL-2.
  • the present invention relates to the identification of monoclonal antibodies or primatized forms thereof which are specific to human B7.1 and/or B7.2 antigen and which are capable of inhibiting the binding of B7.1 to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a
  • CTLA-4 receptor Preferably, these antibodies will induce apoptosis and/or potentiate apoptosis in combination with other agents, e.g. chemotherapy.
  • Blocking of the primary activation site between CD28 and B7.1 (CD80) with the identified antibodies while allowing the combined antagonistic effect on positive co-stimulation with an agnostic effect on negative signaling will be a useful therapeutic approach for intervening in relapsed forms of autoimmune disease.
  • the functional activity of the identified antibodies is defined by blocking the production of the T cell stimulatory cytokine JJ -2.
  • Preparation of monkey monoclonal antibodies will preferably be effected by screening of phage display libraries or by preparation of monkey heterohybridomas using B lymphocytes obtained from B7 [e.g., human B7.1 (CD80) and/or B7.2 (CD86)] immunized monkeys.
  • B7 e.g., human B7.1 (CD80) and/or B7.2 (CD86)
  • the first method for generating anti-B7 antibodies involves recombinant phage display technology. This technique is generally described supra.
  • this vector will comprise synthesis of recombinant immunoglobulin libraries against B7 antigen displayed on the surface of filamentous phage and selection of phage which secrete antibodies having high affinity to B7.1 and/or B7.2 antigen. As noted supra, preferably antibodies will be selected which bind to both human B7.1 and B7.2.
  • PMS This vector, is described in detail infra, and is shown in Figure 1.
  • this vector contains specific primers for PCR amplifying monkey immunoglobulin genes. These primers are based on macaque sequences obtained while developing the primatized technology and databases containing human sequences.
  • Suitable primers are disclosed in commonly assigned U.S. Patent No. 5,658,570.
  • the second method involves the immunization of monkeys, i.e., macaques, against human B7 antigen, preferably against human B7.1 and B7.2 antigen.
  • monkeys i.e., cynomolgus monkeys
  • the resultant antibodies may be used to make primatized antibodies according to the methodology of Newman et al, Biotechnology 10, 1455-1460 (1992), and Newman et al, commonly assigned U.S. Serial No. U.S. Patent No.
  • cynomolgus macaque monkeys are administered human B7 antigen, e.g., human B7.1 (CD80) and/or human B7.2 (CD86) antigen
  • B cells are isolated therefrom, e.g., lymph node biopsies are taken from the animals, and B lymphocytes are then fused with KH6/B5 (mouse x human) heteromyeloma cells using polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Antibodies which bind to both B7.1 and B7.2 are desirable because such antibodies potentially maybe used to inhibit the interaction of B7.1 (CD80) and B7.2 (CD86), as well as B7 with their counter-receptors, i.e., human CTLA-4 and CD28. Antibodies against these epitopes may inhibit the interaction of both human B7.1 (CD80) and human B7.2 (CD86) with their counter receptors on the T cell. This may potentially provide synergistic effects. However, antibodies which bind to only one of human B7 antigen, B7.1 antigen or B7.2 antigen, are also highly desirable because of the co-involvement of these molecules in T cell activation, clonal expansion lymphokine (IL-2) secretion, and responsiveness to antigen.
  • IL-2 clonal expansion lymphokine
  • the disclosed invention involves the use of an animal which is primed to produce a particular antibody.
  • Animals which are useful for such a process include, but are not limited to, the following: mice, rats, guinea pigs, hamsters, monkeys, pigs, goats and rabbits.
  • the present inventors elected to immunize macaques against human B7.1 antigen using recombinant soluble B7.1 antigen produced in CHO cells and purified by affinity chromatography using a L307.4-sepharose affinity column.
  • human B7 antigen human B7.1 (CD80) antigen or human B7.2 (CD86) antigen is not critical, provided that it is of sufficient purity to result in a specific antibody response to the particular administered B7 antigen and potentially to other B7 antigens.
  • human B7 antigen, human B7.1 antigen (CD80) and human B7.2 antigen (CD86) genes have been cloned, and sequenced, and therefore may readily be manufactured by recombinant methods.
  • the administered human B7 antigen, human B7.1 (CD80) antigen and/or human B7.2 (CD86) antigen will be administered in soluble form, e.g., by expression of a B7, B7.1 (CD80) or B7.2 (CD86) gene which has its transmembrane and cytoplasmic domains removed, thereby leaving only the extracellular portion, i.e., the extracellular superfamily V and C-like domains.
  • a B7, B7.1 (CD80) or B7.2 (CD86) gene which has its transmembrane and cytoplasmic domains removed, thereby leaving only the extracellular portion, i.e., the extracellular superfamily V and C-like domains.
  • the macaques will be immunized with the B7antigen, e.g., B7.1 (CD80) and/or B7.2 (CD86) antigen, preferably a soluble form thereof, under conditions which result in the production of antibodies specific thereto.
  • the soluble human B7 antigen e.g., B7.1 (CD80) or B7.2 (CD86) antigen will be administered in combination with an adjuvant, e.g., Complete Freund's Adjuvant (CFA), Alum,
  • CFA Complete Freund's Adjuvant
  • Saponin or other known adjuvants, as well as combinations thereof. In general, this will require repeated immunization, e.g., by repeated injection, over several months. For example, admimstration of soluble B7.1 (CD80) antigen was effected in adjuvant, with booster immunizations, over a 3 to 4 month period, with resultant production of serum containing antibodies which bound human B7.1 (CD80) antigen.
  • B cells are collected, e.g., by lymph node biopsies taken from the immunized animals and B lymphocytes fused with KH6/B5 (mouse x human) heteromyeloma cells using polyethylene glycol. Methods for preparation of such heteromyelomas are known and may be found in U.S. Patent No. 5,658,570, by Newman et al.
  • Heterohybridomas which secrete antibodies which bind human B7 antigen are then identified. This may be effected by known techniques. For example, this may be determined by ELISA or radioimmunoassay using enzyme or radionucleotide labelled human B7 antigen, e.g. B7.1 (CD80) and/or B7.2 (CD86) antigen.
  • B7.1 (CD80) and/or B7.2 (CD86) antigen are then subcloned to monoclonality.
  • the inventors screened purified antibodies for their ability to bind to soluble B7.1 (CD80) antigen coated plates in an ELISA assay, antigen positive B cells, and CHO transfectomas which express human B7.1 (CD80) antigen on their cell surface.
  • the antibodies were screened for their ability to block B cell/T cell interactions as measured by JL-2 production and tritiated thymidine uptake in a mixed lymphocyte reaction (MLR), with B7 binding being detected using 125 I-radiolabeled soluble B7.1 (soluble B7.1 antigen).
  • binding of antibodies to B7.2 (CD86) antigen may be confirmed using soluble B7.2- Ig reagents. As discussed in the examples, this may be effected by producing and purifying B7.2-Ig from CHO transfectomas in sufficient quantities to prepare a B7.2- Ig-sepharose affinity column. Those antibodies which cross-react with B7.2 (CD86) will bind the B7.2-Ig-sepharose column.
  • the cloned monkey variable genes are then inserted into an expression vector which contains human heavy and light chain constant region genes.
  • this is effected using a proprietary expression vector of JJDEC, Inc., referred to as NEOSPLA.
  • This vector is shown in Figure 2 and 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, human immunoglobulin kappa or lambda constant region, the dihydrofolate reductase gene, the human immunoglobulin gamma 1 or gamma 4 PE constant region and leader sequence.
  • This vector has been found to result in very high level expression of primatized antibodies upon incorporation of monkey variable region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
  • this expression system has been previously disclosed to result in primatized antibodies having high avidity (Kd ⁇ 10 "10 M) against CD4 and other human cell surface receptors.
  • the antibodies have been found to exhibit the same affinity, specificity and functional activity as the original monkey antibody.
  • This vector system is substantially disclosed in commonly assigned U.S. Patent No. 5,658,570, incorporated by reference herein, as well as U.S. Serial No. 08/149,099, now U.S. Patent No. 5,736,137, also incorporated by reference in its entirety herein. This system provides for high expression levels, i.e., > 30 pg/cell/day.
  • monkey monoclonal antibodies which specifically bind the B7.1 antigen. These monkey monoclonal antibodies are referred to herein as 7B6, 16C10, 7C10 and 20C9.
  • these antibodies were evaluated for their ability to block B cell/T cell interactions as measured by IL-2 production and tritiated thymidine uptake in a mixed lymphocyte reaction for T cell binding experiments for T cell binding, human buffy coat peripheral blood lymphocytes were cultured for 3-6 days in the presence of PHA stimulator. B7 binding was radioassayed using I-radiolabeled soluble B7.1. The observed results indicate that all of these antibodies bind B7.1 antigen with high affinity and effectively block B cell/T cell interactions as evidenced by reduced IL-2 production and reduced proliferation of mixed lymphocyte cultures.
  • the MLR showed that all 4 anti-B7.1 antibodies inhibit JL-2 production to different extents as shown by the following Ic 5 o values: a: 7B6: 5.0 ⁇ g/M b: 16C10: ⁇ 0.1 ⁇ g/M c: 20C9: 2.0 ⁇ g/M d: 7C10: 5.0 ⁇ g/M
  • the monkey anti-B7.1 antibodies were tested for their ability to bind
  • variable domains of 7C10, 7B6 and 16C10 have cloned the variable domains of 7C10, 7B6 and 16C10, and provide the amino acid and nucleic acid sequences of primatized forms of the 7C10 light chain, 7C10 heavy chain, 7B6 light chain, 7B6 heavy chain, 16C10 light chain and 16C10 heavy chain.
  • amino acid and nucleic acid sequences may be found in Figures 3a and 3b, 4a and 4b, and 5a and 5b.
  • the DNA and amino acid sequence for the human gamma 1, gamma 4 constant domain may be found in U.S. Patent No. 5,658,570.
  • these primatized antibodies are preferably expressed using the NEOSPLA expression vector shown in Figure 2 which is substantially described in commonly assigned U.S. Patent No. 5,658,570, and U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety.
  • the subject primatized antibodies will preferably contain either the human immunoglobulin gamma 1 or gamma 4 constant region, with gamma 4 preferably mutated at two positions to create gamma 4 PE.
  • the gamma 4 PE mutant contains two mutations, a glutamic acid in the CH2 region introduced to eliminate residual FCR binding, and a proline substitution in the hinge region, intended to enhance the stability of the heavy chain disulfide bond interaction. (See, Alegre et al, J Immunol. 148, 3461-3468 (1992); and Angel et al, Mol Immunol. 30, 105-158 (1993), both of which are incorporated by reference herein).
  • the subject primatized antibodies contain the gamma 1, gamma 4 or gamma 4 PE constant region largely depends on the particular disease target.
  • depleting and non-depleting primatized IgGl and IgG4 antibodies are created and tested against specific disease targets.
  • these anti-B7.1 monoclonal antibodies and primatized forms thereof should be well suited as therapeutic agents for blocking the B7:CD28 interaction thereby providing for immunosuppression.
  • these monkey monoclonal antibodies and primatized forms thereof should function as effective immunosuppressants which modulate the B7:CD28 pathway. This is significant for the treatment of many diseases wherein immunosuppression is therapeutically desirable, e.g., autoimmune diseases, to inhibit undesirable antigen specific IgG responses, and also for prevention of organ rejection and graft- versus-host disease.
  • the subject antibodies will be useful in treating any disease wherein suppression of the B7:CD28 pathway is therapeutically desirable.
  • Key therapeutic indications for the subject anti-B7.1 antibodies include, by way of example, autoimmune diseases such as idiopathic thrombocytopema purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, multiple sclerosis, aplastic anemia, psoriasis, allergy, inflammatory bile disease and rheumatoid arthritis.
  • autoimmune diseases such as idiopathic thrombocytopema purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, multiple sclerosis, aplastic anemia, psoriasis, allergy, inflammatory bile disease and rheumatoid arthritis.
  • Another significant therapeutic indication of the subject anti-B7.1 antibodies is for prevention of graft- versus-host-disease (GVHD) during organ transplant and bone marrow transplant (BMT).
  • the subject antibodies may be used to induce host tolerance to donor-specific alloantigens and thereby facilitate engraftment and reduce the incidence of graft rej ection. It has been shown in a murine model of allogeneic cardiac transplantation that intravenous admimstration of CTLA4-Ig can result in immunosuppression or even induction of tolerance to alloantigen. (Lin et al, J. Exp. Med. 178:1801, 1993; Torka et al, Proc. Natl. Acad. Sci., USA 89:11102, 1992). It is expected that the subject primatized anti-B7.1 antibodies will exhibit similar or greater activity.
  • Antibodies produced in the manner described above, or by equivalent techniques can be purified by a combination of affinity and size exclusion chromatography for characterization in functional biological assays. These assays include determination of specificity and binding affinity as well as effector function associated with the expressed isotype, e.g., ADCC, or complement fixation.
  • Such antibodies may be used as passive or active therapeutic agents against a number of human diseases, including B cell lymphoma, infectious diseases including viral diseases such as HIV/AIDS, autoimmune and inflammatory diseases, and transplantation.
  • the antibodies can be used either in their native form, or as part of an antibody/chelate, antibody/drug or antibody/toxin complex. Additionally, whole antibodies or antibody fragments (Fab 2 , Fab, Fv) may be used as imaging reagents or as potential vaccines or immunogens in active immunotherapy for the generation of anti-idiotypic responses.
  • the amount of antibody useful to produce a therapeutic effect can be determined by standard techniques well known to those of ordinary skill in the art.
  • the antibodies will generally be provided by standard technique within a pharmaceutically acceptable buffer, and may be administered by any desired route. Because of the efficacy of the presently claimed antibodies and their tolerance by humans it is possible to administer these antibodies repetitively in order to combat various diseases or disease states within a human.
  • the anti-B7.1 and or B7.2 antibodies (or fragments thereof) of this invention are useful for inducing immunosuppression, i.e., inducing a suppression of a human's or animal's immune system. Also, the invention relates to the use of antibodies specific to B7 antigen, e.g.
  • B7.1 and/or B7.2 antigen for inducing apoptosis of B7 antigen expressing cells, e.g. B cells, and the treatment of any condition wherein apoptosis of B7 antigen expressing cells is therapeutically beneficial.
  • B7 antigen expressing cells e.g. B cells
  • examples of such conditions include those involving B cell tumors and cancers such as B cell lymphomas and leukemias.
  • B cell lymphomas and leukemia suitable for treatment include Hodgkin's disease (all forms, e.g., relapsed Hodgkin's disease, resistant Hodgkin's disease) non-Hodgkin's lymphomas (low grade, intermediate grade, high grade, and other types).
  • Examples include small lymphocytic/B cell chronic lymphocytic leukemia (SLL/B-CLL), lymhoplasmacytoid lymphoma (LPL), mantle cell lymphoma (MCL), foUicular lymphoma (FL), diffuse large cell lymphoma (DLCL), Burkitt's lymphoma (BL), AIDS- related lymphomas, monocytic B cell lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic, foUicular, diffuse large cell, diffuse small cleaved cell, large cell immunoblastic lymphoblastoma, small, non-cleaved, Burkitt's and non-Burkitt's, foUicular, predominantly large cell; foUicular, predominantly small cleaved cell; and foUicular, mixed small cleaved and large cell lymphomas.
  • SLL/B-CLL small lymphocytic/B cell chronic lymphocytic
  • lymphoma classifications include immunocytomal Waldenstrom's MALT-type/monocytoid B cell, mantle cell lymphoma B-CLL/SLL, diffuse large B-cell lymphoma, foUicular lymphoma, and precursor B-LBL.
  • B cell malignancies further include especially leukemias such as ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL), chronic leukocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous leukemia, and promyelocytic leukemia and monocytic cell leukemias.
  • ALL-L3 Breastt's type leukemia
  • CLL chronic lymphocytic leukemia
  • chronic leukocytic leukemia chronic leukocytic leukemia
  • chronic leukocytic leukemia chronic leukocytic leukemia
  • chronic leukocytic leukemia chronic leukocytic leukemia
  • acute myelogenous leukemia acute lymph
  • apoptosis inducing antibodies may be used in conjunction with other treatments that may induce or promote apoptosis such as chemotherapeutics, radioimmunotherapy, and antisense therapy.
  • suitable chemotherapeutics include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustards such as chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine
  • paclitaxel TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ
  • doxetaxel Texotere, Rhone-Poulenc Rorer, Antony, France
  • chiorambucil gemcitabine
  • 6-thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • a particularly preferred chemotherapeutic is adriamycin, as IDEC-114 (anti- B7.1 antibody exemplified herein) potentiates apoptosis of lymphoma cells in combination therewith.
  • This invention therefore relates to a method of prophylactically or therapeutically inducing immunosuppression in a human or other animal in need thereof by administering an effective, non-toxic amount of such an antibody of this invention to such human or other animal.
  • the ability of the compounds of this invention to induce immunosuppression has been demonstrated in standard tests used for this purpose, for example, a mixed lymphocyte reaction test or a test measuring inhibition of T-cell proliferation measured by thymidine uptake.
  • cytokines produced in response to co-stimulatory signals that activate CD4+ T cells.
  • the production and secretion of these cytokines occurs naturally in T cells under conditions where a primary and secondary signal is generated through interactions between T cells and antigen presenting cells. Normally a primary signal is initiated through interaction of a antigen specific T cell receptor and MHC Class II molecules bearing the specific antigen on antigen presenting cells. A secondary or costimulatory signal is required to obtain maximal activation of T cells.
  • T cell co-stimulatory receptors have been identified that drive the production of various cytokines, and up-regulate other cell surface receptors that function in growth and differentiation of T cells and hematopoietic accessory cells.
  • T cell co-stimulatory receptors Some of the known signaling T cell co-stimulatory receptors are CD28, CD11, CD54 and CD40L. Sustained adhesion and prolonged interactions through these cell surface molecules result in secretion of IL-2 and various secondary inflammatory cytokines that control numerous immuno-regulatory functions. The study of T cell interactions can be complex due to the presence of numerous accessory cell types capable of mediating redundant or interdependent co-stimulatory effects.
  • antibodies according to the invention to induce apoptosis is evidenced by in vitro capsase induction assays and may be confirmed by other apoptosis assays.
  • the CD28/B7 receptor ligand interaction is considered to be the key secondary response element between antigen presenting cells and CD4+ helper T cells in the immune response cascade.
  • a primary signal is generated between antigen specific T cell receptors and antigen/MHC class JJ complexes, two types of B7 molecules, B7-1 (CD80) and B7-2, (CD86) are up-regulated and establish a membrane signaling event through binding to CD28 receptor. These signals drive the gene expression of various cytokines beginning with the production of IL-2.
  • the detection of secreted IL-2, cell proliferation and various cell surface activation markers including the receptor for IL-2 are clear indicators that co-stimulation has occurred and cells are beginning to divide and differentiate to maturity.
  • T cells may be influenced or driven down different maturation pathways depending on many complex internal and external factors through mechanisms that are poorly understood.
  • the CD28/B7 interaction was first identified as an adhesion event when a B7 specific antibody was identified that blocked adhesion between B and T cell types.
  • CD28 is known to affect in vivo immune responses by functioning both as a cell adhesion molecule linking B and T lymphocytes and as the surface component of a novel signal transduction pathway (June et al. 1990, Immunology Today, 11: 211-216).
  • monoclonal antibodies that recognize either CD28 or B7 antigen e.g. B7.1 and B7.2, are capable of blocking both adhesion and signaling events. Blocking of either event would lead directly or indirectly to reduced signaling through the CD28 receptor and would result in reduced IL-2 production, proliferation and the appearance of secondary cytokines.
  • the present inventors have isolated certain novel antibodies, the activity of which apparently does not involve directly blocking of signal transduction as demonstrated through the use of CTLA-4Ig, a soluble receptor fusion protein that co-recognizes both B7 receptors.
  • CTLA-4Ig a primatized antibody according to the invention
  • IDEC- 114 blocks adhesion of antigen presenting cells to T cells thereby blocking an upstream event prior to signaling that under certain conditions, possibly related to B7 receptor density, is capable of influencing T cell activation.
  • Evidence is provided through use of an in vitro assay that establishes distinct differences between the mechanism of action of IDEC- 114 and other anti-CD80 antibodies as well as CTLA-4Ig.
  • the in vitro assay employed in these experiments was designed to reduce the number of complex interactions provided by accessory cells, by using a purified CD4+ T cell population and replacing accessory cells with a non-cellular co-stimulatory system.
  • This cell activating system obviates the need for antigen presenting cells by using latex micospheres containing immobilized antibody to the CD3 antigen to deliver a suboptimal primary signal to the T cell.
  • This system when presented along with B7, e.g. B7.1 (CD80) antigen and/or B7.2 (CD86) antigen, co-stimulatory ligand provides a very potent signal through the CD28 receptor that initiates gene expression resulting in production of IL-2, T cell growth and other pro-inflammatory cytokines.
  • the antibodies of this invention have utility in inducing immunosuppression indicates that they should be useful in the treatment or prevention of resistance to or rejection of transplanted organs or tissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.); the treatment or prevention of autoimmune, inflammatory, proliferative and hyperproliferative diseases, and of cutaneous manifestations of immunologically medicated diseases (e.g., rheumatoid arthritis, lupus erythematosus, systemic lupus erythematosus, Hashimotos thyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis, nephrotic syndrome, psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitides, seborrheic dermatitis, Lichen planus, Pemplugus, bullous pemphigus, Epidermolysis bullosa,
  • antibodies according to the invention maybe used in therapies wherein apoptosis of B7 expressing cells is therapeutically beneficial such as cancers characeterized by cancerous B cells, or cancers having an adverse B cell involvement. It has been reported, e.g. that B cells may promote the growth and/or metastasis of some cancers (not characterized by cancerous B cells), including some solid tumor types.
  • the subject antibodies may promote apoptosis of other agents such as chemotherapeutics, antisense oligos, and radiotherapeutics.
  • an effective dosage will be in the range of about 0.05 to 100 milligrams per kilogram body weight per day.
  • the antibodies (or fragments thereof) of this invention should also be useful for treating tumors in a mammal. More specifically, they should be useful for reducing tumor size, inhibiting tumor growth and/or prolonging the survival time of tumor-bearing animals. Accordingly, this invention also relates to a method of treating tumors, e.g. those associated with B cell lymphomas, in a human or other animal by administering to such human or animal an effective, non-toxic amount of an antibody.
  • an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day.
  • the 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.
  • Such 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.
  • the route of administration of the antibody (or fragment thereof) of the invention include, by way of example, oral, parenteral, inhalation and topical.
  • parenteral as used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
  • the subcutaneous and intramuscular forms of parenteral admimstration are generally preferred.
  • the daily parenteral and oral dosage regimens for employing compounds of the invention to prophylactically or therapeutically induce immunosuppression, or to therapeutically treat carcinogenic tumors will generally be in the range of about 0.05 to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.
  • the antibodies of the invention may also be administered by inhalation.
  • inhalation is meant intranasal and oral inhalation administration.
  • Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
  • the preferred dosage amount of a compound of the invention to be employed is generally within the range of about 10 to 100 milligrams.
  • the antibodies of the invention may also be administered topically.
  • topical administration is meant non-systemic administration and includes the application of an antibody (or fragment thereof) compound of the invention externally to the epidermis, to the buccal cavity and instillation of such an antibody into the ear, eye and nose, and where it does not significantly enter the blood stream.
  • systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
  • the amount of an antibody required for therapeutic or prophylactic effect will, of course, vary with the antibody chosen, the nature and severity of the condition being treated and the animal undergoing treatment, and is ultimately at the discretion of the physician.
  • a suitable topical dose of an antibody of the invention will generally be within the range of about 1 to 100 milligrams per kilogram body weight daily.
  • Formulations While it is possible for an antibody or fragment thereof to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • the active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w but preferably not in excess of 5% w/w and more preferably from 0.1 % to 1 % w/w of the formulation.
  • the topical formulations of the present invention comprise an active ingredient together with one or more acceptable carrier(s) therefor and optionally any other therapeutic ingredients(s).
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
  • Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 90°-100°C for half an hour.
  • the solution may be sterilized by filtration and transferred to the container by an aseptic technique.
  • bactericidal and fungicidal agents suitable for inclusion in the drops are phenylrnercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis.
  • the basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.
  • the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
  • the subject anti-B7.1 antibodies or fragments thereof may also be administered in combination with other moieties which modulate the B7:CD28 pathway.
  • moieties include, by way of example, cytokines such as JL-7 and J_L- 10, CTLA4-Ig, soluble CTLA-4 and anti-CD28 antibodies and fragments thereof.
  • the subject antibodies maybe administered in combination with other immunosuppressants.
  • immunosuppressants include small molecules such as cyclosporin A (CSA) and FK506; monoclonal antibodies such as anti-tumor necrosis factor a (anti-TNFa), anti-CD54, anti-CDll, anti-CD 11 a, and anti-E -l; and, other soluble receptors such as rTNFa and rIL-1.
  • CSA cyclosporin A
  • FK506 monoclonal antibodies such as anti-tumor necrosis factor a (anti-TNFa), anti-CD54, anti-CDll, anti-CD 11 a, and anti-E -l
  • anti-TNFa anti-tumor necrosis factor a
  • anti-TNFa anti-tumor necrosis factor a
  • anti-CD54 anti-CD54
  • anti-CDll anti-CD 11 a
  • anti-E -l anti-E -l
  • other soluble receptors such as rTNFa and rIL-1.
  • the optimal quantity and spacing of individual dosages of an antibody or fragment thereof of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular animal being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of an antibody or fragment thereof of the invention given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • a pharmaceutical composition of this invention in the form of a capsule is prepared by filling a standard two-piece hard gelatin capsule with 50 mg. of an antibody or fragment thereof of the invention, in powdered form, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesium stearate.
  • Iniectable Parenteral Composition is prepared by filling a standard two-piece hard gelatin capsule with 50 mg. of an antibody or fragment thereof of the invention, in powdered form, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesium stearate.
  • a pharmaceutical composition of this invention in a form suitable for administration by injection is prepared by stirring 1.5% by weight of an antibody or fragment thereof of the invention in 10% by volume propylene glycol and water. The solution is sterilized by filtration.
  • Antibody or fragment thereof of the invention 1.0 g. White soft paraffin to 100.0 g.
  • the antibody or fragment thereof of the invention is dispersed in a small volume of the vehicle to produce a smooth, homogeneous product. Collapsible metal tubes are then filled with the dispersion.
  • Topical Cream Composition
  • Antibody or fragment thereof of the invention 1.0 g.
  • Topical Lotion Composition The polawax, beeswax and lanolin are heated together at 60°C A solution of methyl hydroxybenzoate is added and homogenization is achieved using high speed stirring. The temperature is then allowed to fall to 50°C The antibody or fragment thereof of the invention is then added and dispersed throughout, and the composition is allowed to cool with slow speed stirring.
  • Antibody or fragment thereof of the invention 1.0 g. Sorbitan Monolaurate 0.6 g.
  • the methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of the water at 75°C
  • the sorbitan monolaurate, polysorbate 20 and cetostearyl alcohol are melted together at 75°C and added to the aqueous solution.
  • the resulting emulsion is homogenized, allowed to cool with continuous stirring and the antibody or fragment thereof of the invention is added as a suspension in the remaining water. The whole suspension is stirred until homogenized.
  • Antibody or fragment thereof of the invention 0.5 g.
  • Purified Water B.P. to 100-00 ml.
  • the methyl and propyl hydroxybenzoates are dissolved in 70 ml. purified water at 75°C and the resulting solution is allowed to cool.
  • the antibody or fragment thereof of the invention is then added, and the solution is sterilized by filtration through a membrane filter (0.022 ⁇ m pore size), and packed aseptically into suitable sterile containers.
  • composition for Administration by Inhalation Composition for Administration by Inhalation
  • an aerosol container with a capacity of 15-20 ml dissolve 10 mg. of an antibody or fragment thereof of the invention in ethanol (6-8 ml.), add 0.1 -0.2% of a lubricating agent, such as polysorbate 85 or oleic acid; and disperse such in a propellant, such as freon, preferably in combination of (1.2 dichlorotetrafluoroethane) and difluorochloromethane, and put into an appropriate aerosol container adapted for either intranasal or oral inhalation administration.
  • a lubricating agent such as polysorbate 85 or oleic acid
  • disperse such in a propellant, such as freon, preferably in combination of (1.2 dichlorotetrafluoroethane) and difluorochloromethane
  • compositions for parenteral administration will commonly comprise a solution of an antibody or fragment thereof of the invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well-known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • the concentration of the antibody or fragment thereof of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight, and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 Ml sterile buffered water, and 50 mg. of an antibody or fragment thereof of the invention.
  • a phannaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml. of sterile Ringer's solution, and 150 mg. of an antibody or fragment thereof of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania, hereby incorporated by reference herein.
  • the antibodies (or fragments thereof) of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconstitution techniques can be employed.
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially anest the disease and its complications.
  • compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Single or multiple administrations of the pharmaceutical compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical composition of the invention should provide a quantity of the altered antibodies (or fragments thereof) of the invention sufficient to effectively treat the patient.
  • antibodies of this invention may be used for the design and synthesis of either peptide or non-peptide compounds (mimetics) which would be useful in the same therapy as the antibody. See, e.g., Saragovi et al., Science, 253, 792-795 (1991).
  • 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 (Mowa 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 HI product which is present in three to five copies at one end of the phage.
  • the gene HI 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 CHI domain, are fused to the carboxy-terminal half of the gene in 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 HJ.
  • the FLAG is an 8 amino acid peptide expressed at the carboxy terminal of the Fd protein.
  • Non-adherent phage were washed off and the adherent phage were eluted with acid. The eluted phage were used to infect a fresh aliquot of XLlBlue bacteria and helper phage was added. After overnight amplification, phage were prepared and again panned on antigen coated plates. After three rounds of panning, we were able to show that we had successfully enriched for the anti-tetanus toxoid phage. The success of this technology also depends on the ability to prepare soluble Fabs for characterization of the final panned product. This was achieved by excising gene HI from the pMS DNA using the restriction enzyme Nhe I followed by re-ligation.
  • Fab was no longer displayed on the phage surface but accumulated in the piroplasmic space. Lysates were prepared from bacteria expressing soluble Fab and tested for antigen specificity using an ELISA. High levels of soluble Fab were detected.
  • the first set of primers was designed for amplification of the heavy chain VH and CHI (Fd) domains. It consisted of a 3' CHI domain primer and six 5' VH family specific primers that bind in the framework 1 region.
  • Our second set of primers for amplifying the whole lambda chain, covers the many lambda chain subgroups. It consists of a 3' primer and three 5' degenerate primers that bind in the VL framework 1 region.
  • Our third set of primers was designed for amplification of the kappa chain subgroups. It consists of one 3' primer and five VK framework 1 primers.
  • RNA was extracted from SB cells and converted to cDNA using reverse transcriptase.
  • the first strand cDNA was PCR amplified using B7.1 specific primers and cloned into IDEC's NEOSPLA mammalian expression vectors.
  • CHO cells were transfected with B7.1 NEOSPLA DNA and clones expressing membrane associated B7.1 were identified.
  • 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/Ig NEOSPLA DNA and stable clones secreting B7.1/Ig fusion protein were amplified, hi general, 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-Ig and IL-4, and for the CTLA4 constructs the gene source was PHA activated human T cells.
  • 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 I/Xba I restriction enzymes, and cloned to create the recombinant library.
  • XLI-Blue 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 HI excised. Soluble Fab fragments are generated and tested in ELISA for binding to B7.1 and B7.2.
  • Phage Fab Fragments The monkey phage Fab fragments are characterized for their specificity and the ability to block B7.1-Ig and B7.2-Ig binding to CTLA-4-Ig 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. BioEng. 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-Ig. Kd values are calculated for each fragment after measurements of direct binding to B7.1-Ig or B7.2-Ig coated plates.
  • Fab fragments most effectively blocking the binding of B7-Ig at the lowest concentrations are selected as lead candidates. Selections are made by competing off 125 I-B7-Ig binding to CTLA-4-Ig 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, J. Exp. Med.
  • MLR mixed lymphocyte reaction
  • 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.
  • 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 (Id.)).
  • 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 (Canol 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 I-B7-Ig 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 H 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 MLR showed that all 4 anti-B7.1 antibodies inhibit IL-2 production to different extents: a: 7B6: 5.0 ⁇ g/Ml b: 16C10: 0.1 ⁇ g/Ml c: 20C9: 2.0 ⁇ g/Ml d: 7C10: 5.0 ⁇ g/Ml
  • 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 Clq binding by FACS analysis. Results showed 7C10 monkey Ig had strong human Clq binding after incubating with B7.1 CHO-transfected cells. 16C10 was also positive, while 20C9 and 7B6 monkey antibodies were negative.
  • 16C10 were cloned and primatized forms thereof have been synthesized in CHO cells using the NEOSPLA vector system.
  • the amino acid and nucleic acid sequences for the primatized 7C10 light and heavy chain, 7B6 light and heavy chain, and 16C10 light and heavy chain are respectively shown in Figures 3a, 3b, 4a, 4b, 5a and 5b.
  • EXAMPLE 10 Comparing the ability ofL307.4 andBB-1 mouse antibodies to bind to B7 CHO cells in the presence of CTLA-4Ig.
  • IDEC- II 4 does not block IL-2 production in co-stimulated T cells.
  • a sub- optimal primary signal was induced by attaching an anti-CD3 antibody and a soluble B7Ig fusion protein to covalently coupled protein-A latex microspheres.
  • a 1:10 ratio of anti-CD3 to B7Ig was used which is a relatively high density of B7 costimulatory molecules that is several times greater than normal cells express based on the relative amounts of IL-2 that are typically produced.
  • Purified CD4+ T cells obtained from blood bank donors and co-cultured the cells in presence or absence of soluble CD28:B7 inhibitors that included anti CD80 antibodies L307.4 and lDEC-114 and soluble CTLA-4Ig fusion protein were added at three concentrations ranging from 10 to 0.
  • IDEC-114 does not block growth in co-stimulated T cells.
  • IDEC-114 partially Modes IL- 10 production in co-stimulated T cells.
  • IDEC-114 blocks IL-2 production in T cells co-stimulated with micro-beads containing reduced amounts ofB7.
  • the ratio of anti-CD3 to B7-Ig was adjusted on the stimulator beads from a 1 : 10 ratio to 8 : 1 or a reduction in B7 of about 80-fold with an 8 -fold excess of anti-CD3.
  • the production of IL-2 under these conditions is significantly reduced to typically less than 1000 pg/mL and is more in line with cultures stimulated with mismatched allotypes or CD80 transfected cells. Under these conditions ( Figure 14), we observed near complete inhibition of IL-2 with CTLA-4Ig consistent with results obtained by beads with an anti-CD3/B7-Ig ratio of 1 : 10.
  • IDEC- 114 An experiment was conducted to evaluate the potential ability of IDEC- 114 to induce apoptosis of B7 antigen expressing cells, particularly SKW cells, a known human B cell lymphoma cell line. Whether apoptosis was induced was evaluated based on capsase levels in the presence or absence of IDEC-114 and a suitable control (inelevant antibody). These experiments showed that IDEC-114 induced the expression of capsase relative to the control. As capsase induction is an indicator of apoptosis, these results suggest that anti-B7 antibodies according to the invention may be used to induce apoptosis of B7 antigen expressing cells, e.g. B cells, and preferably B cells associated with B cell related cancers and lymphomas. Analysis of Results.
  • T cell regulatory properties of IDEC- 114 and CTLA-4Ig were compared in an in vitro co-stimulatory system that includes purified CD4+ helper T cells in the absence of accessory cells.
  • Protein A coated latex microspheres and attached anti-CD3 and B7Ig fusion protein were used.
  • T-cells were incubated with beads that contained a 10-fold excess of B7 there was a strong co-stimulatory response as measured by IL-2, IL-10 and cell growth that was totally blocked by CTLA-4Ig and a commercially available anti-CD80 monoclonal antibody L307.4.
  • IDEC-114 had no effect on IL-2 or cell growth but did partially inhibit IL-10 production.
  • CTLA-4 and L307.4 possess higher affinities to B7 antigen and that increasing the concentration of IDEC-114 in the cultures should result in the same effect.
  • antigen presenting cells that become activated may increase their surface density to the extent that the highly mobile B7 molecules more easily form homodimers.
  • monomeric forms of B7 have extremely low affinity and fast off-rate kinetics (van der Merwe, et al. 1997, J. Exp. Med., 185: 393-403) and that homodimeric forms can have up to 500- fold higher affinity.
  • a higher affinity form would understandably facilitate cluster or patch formation leading to a more stable receptor ligand complex. Consequently, IDEC-114 may bind to a remote site that could restrict the association of neighboring CD 80 molecules and reduce or limit the amount of dimerization effectively limiting the adhesion complex formation.

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Abstract

L'invention concerne l'identification d'anticorps qui sont spécifiques des antigènes B7, par exemple les antigènes humains B7.1 (CD80) et B7.2 (CD86), lesquels favorisent l'apoptose des lymphocytes B7+, de préférence des lymphocytes B. De tels anticorps peuvent inhiber la liaison de B7.1 à un récepteur de CD28 mais ne peuvent pas inhiber la liaison de B7.1 à un récepteur de CTLA-4. A titre d'exemple, deux anticorps, le 16C10 et le 7C10, inhibent également de manière importante la production d'IL-2. Ces anticorps sont utiles dans le traitement de pathologies dans lesquelles l'apoptose des lymphocytes B7+ est souhaitable, par exemple les tumeurs à lymphocytes B, les lymphomes, leucémies ou tumeurs solides ou cancers dans lesquels les lymphocytes B favorisent la croissante tumorale et/ou les métastases, mais dans lesquels ces lymphocytes B ne sont eux-mêmes pas cancéreux.
PCT/US2001/016364 2000-05-22 2001-05-22 Identification d'interactions de liaison unique entre certains anticorps et les antigenes humains co-stimulateurs b7.1 et b7.2 Ceased WO2001089567A1 (fr)

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WO2002060485A3 (fr) * 2001-01-31 2003-06-05 Idec Pharma Corp Anticorps immunoregulateurs et ses utilisations
WO2004076488A1 (fr) * 2003-02-27 2004-09-10 Theravision Gmbh Molecule se liant a cd80 et cd86
EP1565489A4 (fr) * 2002-06-19 2007-04-11 Raven Biotechnologies Inc Cible de surface cellulaire raag10 et famille d'anticorps reconnaissant cette cible
WO2008119071A1 (fr) * 2007-03-28 2008-10-02 Biogen Idec Inc. Modulation du microenvironnement d'une tumeur
US7718774B2 (en) 2006-11-08 2010-05-18 Macrogenics, Inc. TES7 and antibodies that bind thereto
EP2314318A1 (fr) * 2001-01-31 2011-04-27 Biogen Idec Inc. Anticorps CD80 pour utilisation en combinaison avec agents chemothérapeutiques pour le traitement de malignité de cellules B
EP2267027A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Procédé d'administration de polypeptides thérapeutiques et polypeptides correspondants
EP2073827A4 (fr) * 2006-11-03 2012-04-25 Univ Northwestern Therapie de la sclerose en plaque
US8475790B2 (en) 2008-10-06 2013-07-02 Bristol-Myers Squibb Company Combination of CD137 antibody and CTLA-4 antibody for the treatment of proliferative diseases
US8840889B2 (en) 2009-08-13 2014-09-23 The Johns Hopkins University Methods of modulating immune function
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
EP3845652A4 (fr) * 2018-06-22 2022-04-13 JUNTEN BIO Co., Ltd. Anticorps induisant la tolérance immunitaire, lymphocyte induit, et procédé thérapeutique d'agent de thérapie cellulaire utilisant un lymphocyte induit
RU2816592C2 (ru) * 2018-06-22 2024-04-02 ДЗУНТЕН БИО Ко., Лтд. Антитела, индуцирующие иммунную толерантность, индуцированные лимфоциты и терапевтический агент/способ клеточной терапии с использованием индуцированных лимфоцитов

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998019706A1 (fr) * 1996-11-08 1998-05-14 Idec Pharmaceuticals Corporation Identification d'interactions de liaison uniques entre certains anticorps et les antigenes costimulants b7.1 et b7.2 humains

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998019706A1 (fr) * 1996-11-08 1998-05-14 Idec Pharmaceuticals Corporation Identification d'interactions de liaison uniques entre certains anticorps et les antigenes costimulants b7.1 et b7.2 humains

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WO2002060485A3 (fr) * 2001-01-31 2003-06-05 Idec Pharma Corp Anticorps immunoregulateurs et ses utilisations
EP2314318A1 (fr) * 2001-01-31 2011-04-27 Biogen Idec Inc. Anticorps CD80 pour utilisation en combinaison avec agents chemothérapeutiques pour le traitement de malignité de cellules B
US9574007B2 (en) 2002-06-19 2017-02-21 Macrogenics, Inc. B7-H3L cell surface antigen and antibodies that bind thereto
EP2277917A3 (fr) * 2002-06-19 2012-07-11 Raven Biotechnologies, Inc. Cible de surface B7-H3L et famille d'anticorps reconnaissant cette cible
US7527969B2 (en) 2002-06-19 2009-05-05 Raven Biotechnologies, Inc. RAAG10 cell surface target and a family of antibodies recognizing that target
US8779098B2 (en) 2002-06-19 2014-07-15 Macrogenics West, Inc. B7-H3L cell surface antigen and antibodies that bind thereto
EP1565489A4 (fr) * 2002-06-19 2007-04-11 Raven Biotechnologies Inc Cible de surface cellulaire raag10 et famille d'anticorps reconnaissant cette cible
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
EP2267027A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Procédé d'administration de polypeptides thérapeutiques et polypeptides correspondants
US9725522B2 (en) 2002-11-08 2017-08-08 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
WO2004076488A1 (fr) * 2003-02-27 2004-09-10 Theravision Gmbh Molecule se liant a cd80 et cd86
EP2073827A4 (fr) * 2006-11-03 2012-04-25 Univ Northwestern Therapie de la sclerose en plaque
US8216570B2 (en) 2006-11-08 2012-07-10 Macrogenics, Inc. TES7 and antibodies that bind thereto
US7718774B2 (en) 2006-11-08 2010-05-18 Macrogenics, Inc. TES7 and antibodies that bind thereto
JP2010522772A (ja) * 2007-03-28 2010-07-08 バイオジェン・アイデック・インコーポレイテッド 腫瘍微小環境の調整
WO2008119071A1 (fr) * 2007-03-28 2008-10-02 Biogen Idec Inc. Modulation du microenvironnement d'une tumeur
US8475790B2 (en) 2008-10-06 2013-07-02 Bristol-Myers Squibb Company Combination of CD137 antibody and CTLA-4 antibody for the treatment of proliferative diseases
US8840889B2 (en) 2009-08-13 2014-09-23 The Johns Hopkins University Methods of modulating immune function
US9676856B2 (en) 2009-08-13 2017-06-13 The Johns Hopkins University Methods of modulating immune function
EP3845652A4 (fr) * 2018-06-22 2022-04-13 JUNTEN BIO Co., Ltd. Anticorps induisant la tolérance immunitaire, lymphocyte induit, et procédé thérapeutique d'agent de thérapie cellulaire utilisant un lymphocyte induit
RU2816592C2 (ru) * 2018-06-22 2024-04-02 ДЗУНТЕН БИО Ко., Лтд. Антитела, индуцирующие иммунную толерантность, индуцированные лимфоциты и терапевтический агент/способ клеточной терапии с использованием индуцированных лимфоцитов
TWI854983B (zh) 2018-06-22 2024-09-11 日商順天生化股份有限公司 誘導免疫耐受之抗體、被誘導之淋巴球、及使用被誘導之淋巴球之細胞治療劑治療方法
US12139537B2 (en) 2018-06-22 2024-11-12 Junten Bio Co., Ltd. Antibody inducing immune tolerance, induced lymphocyte, and cell therapy agent therapeutic method using induced lymphocyte
IL279653B1 (en) * 2018-06-22 2025-11-01 Junten Bio Co Ltd Antibody inducing immune tolerance, induced lymphocyte, and cell therapy agent therapeutic method using induced lymphocyte

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