Human Monoclonal Antibodies to Fc Alpha Receptor (CD89)

专利摘要:
The present invention discloses human monoclonal antibodies that specifically bind to Fc alpha receptors (CD89), including monoclonal antibodies that specifically respond to Fc receptors on IgA of human effector cells. Binding agents (eg, antibodies) are useful for targeting human effector cells (eg, macrophages) against target cells (eg, cancer cells, infectious agents, etc.). For this purpose, bifunctional antibodies or heterologous antibodies can be constructed that have binding regions derived from anti-Fc-alpha receptor antibodies and binding regions of target-specific antibodies. Targeted effector cells can specifically lyse target cells.
公开号:KR20040062873A
申请号:KR10-2003-7010590
申请日:2002-02-11
公开日:2004-07-09
发明作者:데브라 허드슨；잔 반디윈클；마르쿠스 에이. 반디크
申请人:메다렉스, 인코포레이티드；
IPC主号:

专利说明:

Human Monoclonal Antibodies to Fc Alpha Receptor (CD89)}
[1] Related Applications
[2] This application is incorporated herein by reference under US Provisional Application No. 60 / 268,075, filed February 12, 2001, and US Provisional Application No. 60 / 338,956, filed November 5, 2001, the entirety of which is incorporated herein by reference. Claiming priority)
[3] Receptors for the Fc portion of immunoglobulins are important for triggering many protective functions of monocytes, macrophages and polymorphonuclear cells. The receptors for IgG on these cells (Fcγ receptors or FcγRs) have been extensively studied and have been shown to generate monoclonal antibodies against these receptors and that they are therapeutically effective (eg, the name of the invention is “human See European Patent No. 255 249, "Monoclonal Antibodies to Fc Receptor for Immunoglobulin G on Human Mononuclear Phagocytes" on monocyte phagocytes.
[4] In addition, IgA receptors (Fcα receptors or CD89) can enhance the function of effector cells. The binding of ligand to CD89 triggers phagocytosis and antibody-mediated cytotoxicity of leukocytes and CD89-bearing cell lines. In addition, CD89 can cooperate with receptors for IgG on effector cells to enhance phagocytosis on target cells.
[5] CD89 is a receptor that binds to the Fc portion of IgA, the most abundant Ig in the human body (Kerr, M. A. 1990, Biochem. J. 271: 285-296). CD89 is always expressed on cytotoxic immune effector cells, mainly polymorphonuclear leukocytes (PMN), monocytes, macrophages, neutrophils and eosinophils (Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423). CD89 is also expressed on lymphocyte subpopulations (Morton, HC, et al., 1996, Critical Reviews in Immunology 16: 423) and also on glomerular mesial cells (Gomez-Guerrero, C., et al. , 1996, J. Immunol. 156: 4369-4376). Moreover, the expression of CD89 on monocytes and PMNs was determined by TNF-α (Gesl, A., et al., 1994, Scad. J. Immumol. 39: 151-156; Hostoffer, RW, et al., 1994, The J. Infectious Diseases 170: 82-87), IL-1, GM-CSF, LPS or phorbol esters (Shen L., et al., J. Immunol. 152: 4080-4086; Schiller, CA et al., 1994, Immunology, 81: 598-604), while IFN-γ and TGF-β1 reduce the expression of FcαRI (Reterink, TJF, et al., 1996, Clin. Exp. Immunol. 103: 161-166).
[6] The α-chain of human CD89 is a type I transmembrane molecule that belongs to the Ig super-gene family, which is highly glycosylated and also contains receptors for IgG and IgE. One gene located on chromosome 19 encodes a number of differently spliced isotypes of the FcαRI alpha chain (55-110 kDa; Morton, HC, et al., 1996, Critical Reviews in Immunology 16: 423). Myeloid CD89 has been found to be associated with the FcR γ-chain, which plays a role in CD89 signal transduction (Morton, HC et al. 1995, J. Biol. Chem. 270: 29781; Pfefferkorn, LC, et al. 1995, J. Immunol. 153: 3228-3236; Saito, K. et al., 1995, J. Allergy Clin. Immunol. 96: 1152).
[7] CD89 binds to both antigen bound IgA1 and IgA2, and monomeric IgA1 and IgA2 (Mazangera, RL et al., 1990, Biochem. J. 272: 159-165), in which FcγR and FcεRI are IgG and IgE, respectively. In the same way as saturated with, the receptor is in vivo consistent with being saturated with monomeric IgA antigen. Cross-linking of CD89 on myeloid effector cells with mAbs specific for the polymerizable IgA, IgA immune complex, or epitope inside or outside the ligand binding domain results in degranulation, peroxide release, inflammatory cytokine secretion, and endocytosis. And stimulate phagocytosis (Patty, C., A. Herbelin, A. Lihuen, JF Bach, and RC Monteiro, 1995, Immunology 86: 1-5; Stewart, WW, RL Maz Yegera, L. Shen, and MA Kerr, 1994, J. Leucocyte Biology 56: 481-487; Stewart, WW, and MA Kerr, 1990, Immunology 71: 328-334; Shen, L., 1992, J. Leukocyte Biology 51: 373-378). This physiological response triggered by CD89 may be important for humoral defense of the first gland on mucosal surfaces (Morton, HC, M. van Egmond, and JGJ van de Winkel, 1996, Critical Reviews in Immunology 16: 423). .
[8] Summary of the Invention
[9] The present invention provides an improved immunotherapeutic for exploiting the therapeutic capacity of human CD89, a cytotoxic triggering molecule. Specifically, the present invention provides isolated human monoclonal antibodies that bind to human CD89, as well as therapeutic compositions, bispecific antibodies and molecular complexes containing such antibodies.
[10] In specific embodiments of the invention, the antibody is not inhibited by IgA (eg, the antibody binds to a site different from the IgA binding site on CD89). In other embodiments, the antibody inhibits binding of IgA to CD89 (eg, the antibody binds to or near a binding site of IgA on CD89).
[11] In other specific embodiments of the invention, the antibody has the additional advantage of not activating complement in vivo (eg, does not induce complement-mediated target cell lysis), which has adverse side effects during treatment. Decrease. In another embodiment, the antibody triggers one or more Fc receptor-mediated effector cell activities, such as phagocytosis or peroxide anion release.
[12] In other specific embodiments of the invention, the antibody is an IgG1 (eg, IgG1κ) antibody (eg, an antibody with an IgG1 heavy chain and a kappa light chain). Also included in the present invention are other isotype antibodies, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD and IgE. These antibodies may be complete antibodies or antigen-binding fragments of antibodies, including Fab, F (ab ') ₂ , Fv and Fv chain fragments.
[13] In other specific embodiments of the invention, the antibody is directed to a nucleic acid of human IgG heavy chain and human kappa light chain, comprising the nucleotide sequence shown in SEQ ID NO: 1 or 5, and the conservative sequence variant thereof, respectively, in the variable region; Is coded by In other embodiments, human antibodies include variable regions of IgG heavy and kappa light chains comprising the amino acid sequences shown in SEQ ID NO: 2 or 6 and SEQ ID NOs: 4 or 8, and conservative sequence variants thereof.
[14] Specific antibodies of the invention include, for example, human monoclonal antibodies (mAb) 14.1, 7.4 and 8.2 (also termed 14A8, 7F12 and 8D2, respectively), or epitopes identical to the epitope to which antibodies 14.1, 7.4 or 8.2 bind. Antibodies are included that bind (eg, compete with each other) or have the same functional binding properties as the antibodies.
[15] Human antibodies of the invention are produced recombinantly in host cells (eg, CHO cells or lymphocytic cells), or hybridomas expressing the antibody (ie, human heavy chain trans that encodes the antibody). Directly from hybridomas, including B cells obtained from non-human transgenic animals (eg, transgenic mice) with genomes comprising transgenes and human light chain transgenes and fused with endogenous proliferative cells Can be.
[16] In other specific embodiments of the invention, the human antibodies of the invention may be characterized by one or more of the following properties:
[17] (a) binding specificity for human CD89;
[18] (b) the binding equilibrium binding constant (K _a ) for human CD89 is at least about 10 ⁷ M ⁻¹ ;
[19] (c) the dissociation constant (K _d ) from human CD89 is less than or equal to about 10 ⁻⁸ S ⁻¹ ;
[20] (c) does not activate complement upon binding to CD89 in vivo;
[21] (d) the antibody binds to a site on human CD89 without inhibiting human IgA binding to human CD89;
[22] (e) the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6 and conservative sequence variants thereof, and an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8 and conservative sequence variants thereof Contains light chains.
[23] In another aspect, the invention provides nucleic acid molecules encoding human monoclonal antibodies or antigen binding portions thereof. In addition, recombinant expression vectors (eg, expression vectors comprising the light and heavy chain variable and constant regions of mAb 14.1, 7.4 or 8.2) and nucleic acid encoding the antibodies of the invention, and such vectors Transfected host cells are included in the present invention as a method of producing the antibodies of the present invention by culturing such host cells.
[24] In another aspect, the invention provides an isolated B cell obtained from a transgenic animal other than a human (eg, a transgenic mouse), which expresses a human anti-CD89 antibody of the invention. Preferably, the isolated B cells are from a transgenic animal other than human (eg, a transgenic mouse) immunized with a purified CD89 antigen preparation or a CD89 antigen-rich preparation, and / or cells expressing CD89. To obtain. Preferably, said non-human transgenic animal (eg, transgenic mouse) has a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or part of the antibodies of the invention. The isolated B cells then have endogenous proliferation, becoming a source of human anti-CD89 antibodies (eg, hybridomas).
[25] The present invention therefore also provides hybridomas capable of producing the human monoclonal antibodies of the invention that specifically bind to CD89. In one embodiment, the hybridoma comprises a non-human transgenic animal (eg, a transgenic mouse) having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the antibodies of the invention. B cells obtained from) and fused with infinite proliferation cells. Specific hybridomas of the present invention include 14.1, 7.4 and 8.2.
[26] In another aspect, the invention provides a transgenic animal other than human, such as a transgenic mouse (also referred to herein as "HuMab"), which expresses a human monoclonal antibody that specifically binds to CD89 do. In one specific embodiment, said non-human transgenic animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene that encodes all or part of an antibody of the invention. Such transgenic animals other than humans may be immunized with purified CD89 antigen preparations or preparations rich in CD89 antigens, and / or cells expressing CD89. Preferably, the non-human transgenic animal (eg, transgenic mouse) is a plurality of human monoclonal antibody isotypes (eg IgG) against CD89 by VDJ recombination and isotype switching. , IgA and / or IgM). Isotype conversion can occur, for example, with classical or non-classical isotype conversion.
[27] In another aspect, the present invention provides a method of producing human monoclonal antibodies that specifically react with CD89. In one embodiment, the method comprises purifying a non-human transgenic animal (eg, a transgenic mouse) having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the antibodies of the invention. Immunization with a CD89 antigen preparation or an agent rich in CD89 antigen, and / or cells expressing CD89. The animal's B cells (e.g., spleen B cells) are then obtained and fused with myeloma cells to produce endogenous hybridoma cells that secrete human monoclonal antibodies against CD89.
[28] In another aspect, the human anti-CD89 antibodies of the invention are derivatized or linked to other functional molecules such as other peptides or proteins (eg, antibody fragments such as antibodies, or Fab 'fragments), or Expressed with the molecule. For example, an antibody or antigen-binding portion of the invention may be one or more other molecules, such as other antibodies, cytotoxins, ligands or antigens of cells (eg, to produce bispecific or multispecific antibodies). Functionally (eg, by chemical coupling, gene fusion, non-covalent bond, etc.). Bispecific antibodies and multispecific antibodies of the invention can be used, for example, to express CD89 expressing cells (eg, to select antigens such as epitopes on tumor cells, autoantibody producing cells, pathogen infected cells or any other undesirable cells). For example, it is useful for targeting effector cells), thereby causing cytolysis or phagocytosis of cells or pathogens associated with the antigen. Other target antigens include soluble antigens or complexes of antigens and microorganisms (eg, viruses, parasites and bacteria).
[29] Multispecific molecules of the invention also include trispecific, tetraspecific and other multispecific molecules. In one embodiment, multispecific molecules include anti-enhancement factor (EF) moieties, such as molecules that bind to surface proteins involved in cytotoxic activity.
[30] Accordingly, the present invention encompasses a wide variety of antibody conjugates, bi- and multispecific molecules and fusion proteins, which bind to CD89 expressing cells to target other molecules to or bind to CD89. Target the molecule to another molecule or cell.
[31] In another aspect, the invention provides a human anti-CD89 antibody of the invention that is linked to a therapeutic moiety (eg, a cytotoxic agent, an enzymatically active toxin or fragment thereof, a radioisotope, or a small molecule anticancer agent). It provides a conjugate comprising.
[32] Alternatively, the antibodies of the invention can be administered with them without being linked to the therapeutic and cytotoxic agents. The antibody may be administered concurrently with such an agent (eg, in a single composition or separately) or may be administered before or after administration of the agent. Such agents include cytokines such as G-CSF, GM-CSF, IL-2 or IFN-alpha, and doxorubicin (Adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxide Chemotherapeutic agents such as roxyurea may be included. In addition, the human antibodies of the invention may be administered in conjunction with radiation therapy.
[33] In another aspect, the invention provides a composition comprising a pharmaceutically acceptable carrier and at least one human monoclonal antibody or antigen-binding portion thereof of the invention that specifically binds to CD89 (eg, pharmaceutical and Diagnostic composition / kit). In one embodiment, the composition comprises a combination of human antibodies or antigen-binding portions thereof, preferably each of which binds to a separate epitope. Compositions (eg, pharmaceutical compositions) comprising one or more human monoclonal antibodies of the invention or combinations of antigen-binding portions thereof, and one or more bispecific or multispecific molecules of the invention are also described herein. It is within the scope of the invention.
[34] For use in vivo treatment and prophylaxis of diseases associated with CD89 expression (eg, overexpression), the human antibodies of the present invention can be used in any suitable route of administration (eg, injection and antibody-based known in the art). Different therapeutic routes of clinical products) to a patient (eg, a human subject) in a therapeutically effective amount.
[35] In addition, the human antibodies of the invention can be used to modulate CD89 levels on effector cells, for example by capping and removing receptors on the cell surface. In addition, mixtures of anti-Fc receptors can also be used for this purpose.
[36] In another embodiment, a human antibody of the invention that blocks or inhibits IgA binding to CD89 is a disease characterized by precipitation of circulating IgA-containing complexes and / or IgA-immune complexes (eg, Useful for treating chronic hepatitis, Henoch-Schonlein purpura (HSP), IgA nephropathy (Berger's disease) or IgA-glomerulonephritis. The human antibody of the present invention may be administered to a patient suffering from said disease in order to inhibit or downregulate endogenous IgA binding to CD89 in vivo.
[37] Based on the ability to bind both immune cells with CD89 and specific target cells (ie, cells that may be beneficial to the host if removed), bispecific and multispecific molecules of the invention treat a variety of diseases It can be used to. These diseases include autoimmune diseases and cancers (eg, bladder cancer, breast cancer, colon cancer, kidney cancer, ovarian cancer, testicular cancer, prostate cancer, lung cancer, brain cancer, rectal cancer, pancreatic cancer, liver cancer, central nervous system cancer, head and neck cancer, kidney cancer, Bone cancer, hematologic cancer and lymphatic system cancer); Viral (eg, HIV, HTLV, and FELV) infections, protozoa (eg, Toxoplasma gondii ) infections, fungi (eg, Candida albicans ) infections, and bacteria ( Examples include pathogenic infections such as Staphylococcus aureus , Staphylococcus amorus , and Streptococcus hemolyticus , and Mycobacterium tuberculosis infections. This is not restrictive. Another aspect of the invention relates to vaccination against diseases and cancers by including antigens from diseased organisms, antigens from infected cells, antigens from gene products of the diseased organisms or antigens from cancer cells. Provide useful molecules. For this purpose, the present invention provides a composition as a binding determinant which binds this antigen with a binding determinant which directs a useful effective antigen into the immune system.
[38] In one embodiment, the patient modulates (eg, increases or inhibits) the chemotherapy, radiation, or expression or activity of an Fc receptor (eg, an Fcα receptor or an Fcγ receptor) (eg, , Cytokines). Typical cytokines that can be administered during treatment include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factor (TNF). Included. Typical therapeutic agents include, in particular, anti-tumor agents (eg doxorubicin (Adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea).
[39] In another aspect, the present invention provides a method for detecting the presence of CD89 in a sample in vitro or in vivo (eg, for diagnosing a CD89-related disease). In one embodiment, this is accomplished by contacting a sample to be tested with a human monoclonal antibody (or antigen-binding portion thereof) of the invention under conditions that may form a complex between the antibody and CD89, optionally together with a control sample. Is achieved. Complex formation is then detected (eg using ELISA). When using a control sample with a test sample, complexes are detected in both samples, and all statistically significant differences between samples in complex formation are indicative of the presence of CD89 in the test sample.
[40] Other aspects and advantages of the invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference as if the contents were individually listed.
[41] 1 shows the nucleotide sequence and corresponding amino acid sequence of the V _H -region of HuMAb 14.1. CDR regions are also shown. Germline segments used in the V _H -region of HuMAb 14.1 include V _H 3-30.3, D 6-13 and JH4b.
[42] 2 shows the nucleotide sequence and corresponding amino acid sequence of the V _κ -region of HuMAb 14.1. CDR regions are also shown. Germline cell segments used in the V _κ -region of HuMAb 14.1 include V _κ L18 and JK3.
[43] Figure 3 shows the nucleotide sequence of the V _H -region of HuMAb 8D2 and the corresponding amino acid sequence. CDR regions are also shown. Germline cell segments used in the V _H -region of HuMAb 8D2 include V _H 3-30.3, D 7-27 and JH3b.
[44] 4 shows the nucleotide sequence and corresponding amino acid sequence of the V _κ -region of HuMAb 8D2. CDR regions are also shown. Germline cell segments used in the V _κ -region of HuMAb 8D2 include V _κ A27 and JK2.
[45] The present invention provides isolated human monoclonal antibodies, including antigen-binding portions of isolated human monoclonal antibodies, which bind to epitopes present on the human IgA receptor (CD89). In one embodiment, the human antibody is non-human capable of producing multiple isotypes of human monoclonal antibodies (eg, IgG, IgA and / or IgE) against CD89 via VDJ recombination and isotype conversion. Produced in transgenic animals (eg, transgenic mice). Thus, specific aspects of the present invention include antibodies, antibody fragments, and pharmaceutical compositions thereof, as well as transgenic animals, B-cells, and hybridomas other than humans that produce monoclonal antibodies. In addition, the present invention detects cells expressing CD89 in vitro or in vivo, or triggers effector functions in cells expressing CD89 (e.g., bispecific including the anti-CD89 antibodies of the present invention). Methods of using the antibodies of the invention to crosslink CD89 molecules using antibodies, and other antibodies against antigens on target cells. Methods of using the antibodies of the invention to block or inhibit IgA binding to CD89 are also provided, which are diseases characterized by precipitation of circulating IgA-containing complexes and / or IgA-immune complexes (eg, , Chronic hepatitis, Hennoch-Scholine purpura (HSP), IgA nephropathy (Burgers disease) or IgA-glomerulonephritis) are useful for treating diseases characterized by abnormal endogenous IgA.
[46] In order to make the present invention easier to understand, some terms are first defined. Further definitions have been set forth throughout the detailed description of the invention.
[47] As used herein, the terms "CD89", "human IgA receptor" and "Fc-alpha receptor" (FcαRI) are used interchangeably and are genes of one α-gene (FcαRI) located on chromosome 19. It includes the product. This gene is known to encode a transmembrane isotype of 55 to 110 kDa spliced in many different ways. FcαRI (CD89) is always expressed on monocytes / macrophages, eosinophils and neutrophil granulocytes, but not on non-effector cell populations. FcαRI has a moderate affinity for IgA1 and IgA2 (about 5 x 10 ⁷ M ^-1 ), which increases when exposed to cytokines such as G-CSF or GM-CSF (Morton, HC et al (1996) Critical Reviews in Immunology 16: 423-440). FcαRI receptors are (1) expressed primarily on immune effector cells (eg, monocytes, macrophages, neutrophils and eosinophils); (2) have high expression levels (eg 5,000 to 100,000 per cell); (3) is a mediator of cytotoxic activity (eg ADCC, phagocytosis); (4) It is a preferred triggering receptor for use in the present invention because it mediates enhanced antigen presentation to antigens, including autoantigens.
[48] As used herein, the term “effector cell” means an immune cell that participates in the stage of action of the immune response, as opposed to the stage of recognition and activation of the immune response. Representative immune cells include cells of bone marrow or lymph origin, such as lymphocytes (eg, B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes. , Eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In a preferred embodiment, effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC) (eg, neutrophils can induce ADCC). For example, monocytes, macrophages, expressing FcRs are specifically involved in killing target cells, presenting antigen to other components of the immune system, or binding antigen presenting cells. In other embodiments, effector cells may be phagocytosis against a target antigen, target cell or microorganism. Expression of certain FcRs on effector cells can be regulated by humoral factors such as cytokines. For example, expression of FcαRI has been found to be upregulated by G-CSF or GM-CSF. This increase in expression increases the effector function of the FcαRI-bearing cells on the target. Effector cells may phagocytize or lyse the target antigen or target cells.
[49] “Target cells” are undesirable in subjects (eg, humans or animals) that may be targeted by a composition of the invention (eg, human monoclonal antibodies, bispecific or multispecific molecules). Means any cell. In one embodiment, the target cell is a cell that expresses or overexpresses CD89. In other embodiments, target cells include tumor cells. Tumor cells that may be targeted are any type, including breast cancer, ovarian cancer, prostate cancer, testicular cancer, lung cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, central nervous system cancer, kidney cancer, head cancer, cervical cancer, bone cancer, blood cancer, and lymphatic cancer. Tumor cells in cancer. In addition to tumor cells, the effector cells may be targeted to autoantibody producing lymphocytes to treat autoimmune diseases or to IgE-producing lymphocytes to treat allergies. In addition, the target may be a microorganism (bacteria or virus) or a soluble antigen (eg, rheumatoid like factor, or other autoantibodies and toxins). Microorganisms include pathogens (eg viruses, bacteria, fungi, protozoa).
[50] The term “antigen” refers to any natural immunogenic or synthetic immunogenic material, fragment or portion of an immunogenic material, peptide epitope or hapten. The term "antigen" also includes substances which are non-immunogenic in non-complexed form, but which become immunogenic in complexing. The term “uncomplexed” includes materials prior to being linked to form the molecular complexes of the present invention. The term "complexation" includes materials that are linked to form the molecular complexes of the present invention.
[51] As used herein, the term “inhibition of growth” (eg, when referring to a cell) refers to the cell as compared to cell growth when the cell is not contacted with an anti-CD89 antibody. All measurable cell growth reductions when in contact with (eg, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or Cell growth inhibition of at least 100%).
[52] As used herein, the terms "inhibition of binding" and "binding of blocking" (meaning, for example, inhibiting / blocking the binding of CD89 ligands such as IgA to CD89) are used interchangeably, partially and Complete inhibition / blocking. Inhibition / blocking of IgA to CD89 preferably reduces or alters the normal level or type of effector cell function that occurs when IgA binds to CD89 without being inhibited or blocked. All measurable binding affinity reductions of IgA for CD89 when contacted with an anti-CD89 antibody are included as compared to when not in contact with an anti-CD89 antibody (eg, about 10 CD89 ligands for CD89). %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% or more).
[53] As used herein, the term “antibody” includes complete antibodies and antigen binding fragments (ie, “antigen-binding portions”) or single chains thereof. "Antibody" means a glycoprotein comprising two or more heavy chains (H) and two or more light chains (L), or antigen binding portions thereof, bound to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain constant region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariability regions, termed complementarity determining regions (CDRs), and more highly conserved regions, termed framework regions (FR), between the hypervariable regions. This exists. Each of VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains have binding domains that interact with the antigen. The constant region of the antibody may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of a typical complement system.
[54] As used herein, the term “antigen binding portion” (or simply “antibody portion”) of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, CD89). do. It has been found that the antigen-binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include (i) monovalent Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) a bivalent F (ab ') ₂ segment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of a single arm of the VL and VH domains of an antibody; (v) dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); And (vi) isolated complementarity determining regions (CDRs). In addition, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, a single protein chain (single) is formed using a recombinant method whereby the two domains are paired to form a monovalent molecule in which the VL and VH regions are paired. Known as chain Fv (scFv); see, eg, Bird et al., (1998) Science 242: 423-426 and Houston et al., (1998) Proc. Natl. Acad. Sci. USA 85 (See 5879-5883). Such single chain antibodies are also included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and, depending on the application, the fragments are screened in the same manner as in the complete antibody.
[55] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of a group of chemically active surface molecules such as amino acids or sugar side chains and generally have specific three dimensional structural characteristics as well as specific charging characteristics. In the presence of a modified solvent, structural epitopes and non-structural epitopes are distinguished from one another in that the binding to the structural epitopes is broken but the binding to the non-structural epitopes is not broken.
[56] The term “bispecific molecule” includes any agent having two different binding specificities, such as a protein, peptide, or protein or peptide complex. For example, the molecule may bind to or interact with (a) cell surface antigens and (b) Fc receptors (eg, CD89) on the surface of effector cells. The term “multispecific molecule” or “heterospecific molecule” includes any agent having two or more different binding specificities, such as a protein, peptide, or protein or peptide complex. For example, the molecule may bind to or interact with (a) cell surface antigens, (b) Fc receptors on the surface of effector cells, and (c) one or more other components. Thus, the present invention includes, but is not limited to, bispecific, trispecific, tetraspecific and other multispecific molecules for cell surface antigens such as CD89, and other targets such as Fc receptors on effector cells.
[57] The term “bispecific antibody” further includes diabodies. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but by using short linkers that cannot link between the two domains on the same chain, Pairing results in two antigen binding sites (see, eg, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et. al. (1994) Structure 2: 1121-1123).
[58] As used herein, the term “heterologous antibody” refers to two or more antibodies, antibody binding fragments (eg, Fabs), derivatives thereof, or antigen binding regions linked to one another (two or more of which are different from each other) Specificity). Such different specificities include binding specificity for Fc receptors on effector cells, and binding specificity for antigens or epitopes on target cells (eg, tumor cells).
[59] As used herein, the term “human antibody” includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention include amino acid residues that are not coated by human germline immunoglobulin sequences (e.g., mutations introduced by random or location-specific mutations in vitro or by somatic mutations in vivo). May be included. However, as used herein, the term “human antibody” does not include antibodies in which CDR sequences derived from germline lines of other mammalian species, such as mice, have been implanted into human framework sequences.
[60] As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to an antibody molecule preparation of single molecule composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for specific epitopes. Thus, the term “human monoclonal antibody” refers to an antibody exhibiting single binding specificities, with variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is obtained from a non-human transgenic animal (eg, a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene and fused with endogenous proliferating cells. Produced by hybridomas, including B cells.
[61] As used herein, the term “recombinant human antibody” refers to any human antibody produced, expressed, produced or isolated by recombinant methods, such as (a) the human immunoglobulin gene (described in more detail in paragraph I below). Antibody isolated from a transgenic animal (eg, a mouse), (b) an antibody expressed using a recombinant expression vector transfected into a host cell, (c) an antibody isolated from a recombinant human antibody library, and (d) Included are antibodies prepared, expressed, produced or isolated by any means including splicing a human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments such recombinant human antibodies can be mutated in vitro (or somatically mutated in vivo when using a transgenic animal with a human Ig sequence), and thus, the VH and VL regions of the recombinant antibody. The amino acid sequence is derived from and related to human germline VH and VL sequences, but may not be naturally present in the human germline repertoire in vivo.
[62] As used herein, “heterologous antibody” is defined in the context of non-human transgenic organisms that produce such antibodies. The term refers to an antibody having an amino acid sequence, or coding nucleic acid sequence, corresponding to a sequence found in an organism that does not comprise a transgenic animal other than human, and generally does not consist of a transgenic animal species other than human.
[63] As used herein, the term “heterohybrid antibody” refers to an antibody having light and heavy chains of other organism origin. For example, an antibody having a human heavy chain linked to a murine light chain is a heterologous hybrid antibody. Examples of heterologous hybrid antibodies include the chimeric antibodies and humanized antibodies described above.
[64] As used herein, the term “isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigen specificities (eg, substantially free of antibodies that specifically bind to antigens other than CD89). Isolated antibody that specifically binds). However, isolated antibodies that specifically bind to epitopes, isotypes or variants of human CD89 may have cross reactivity with other related antigens, eg, from other species (eg, CD89 species homologs). Can be. Moreover, the isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the invention, the combination of “isolated” monoclonal antibodies having different specificities are combined in a well defined composition.
[65] As used herein, the term “specific binding” means that the antibody binds to a given antigen. Typically, the antibody binds with an affinity of about 1 × 10 ⁷ M ⁻¹ and binds to non-specific antigens (eg, BSA, casein) other than a predetermined or highly relevant antigen. Binds to the predetermined antigen with affinity higher than 2 times higher. The phrases "antibodies that recognize antigens" and "antibodies specific for antigens" are used interchangeably herein with the terms "antibodies that specifically bind antigens."
[66] As used herein, the term “high affinity” for IgG antibodies is about 10 ⁷ M ⁻¹ or greater, preferably about 10 ⁸ M ⁻¹ or greater, more preferably about 10 ⁹ M ⁻¹ , 10 ¹⁰ A binding affinity of M ⁻¹ , 10 ¹¹ M ⁻¹ or more (eg, 10 ¹³ M ⁻¹ or less, or more). However, "high affinity" binding may vary for different antibody isotypes. For example, “high affinity” binding to an IgM isotype means a binding affinity of at least about 1 × 10 ⁷ M ⁻¹ .
[67] As used herein, the term "K _assoc " or "K _a " refers to the binding constant of a particular antibody-antigen interaction.
[68] As used herein, the term “K _dis ” or “K _d ” refers to the dissociation constant of a particular antibody-antigen interaction.
[69] As used herein, the term “isotype” refers to an antibody class (eg, IgM or IgG1) that is encoded by the heavy chain constant region gene.
[70] As used herein, the term “isotype conversion” refers to the phenomenon in which a class or isotype of an antibody is changed from one Ig class to one class of another Ig class.
[71] As used herein, the term “unconverted isotype” refers to the isotype class of the heavy chain produced when no isotype conversion has occurred, and the CH gene encoding the unconverted isotype is typically functional. It is the first CH gene located directly below the VDJ gene rearranged into. Isotype conversions are classified as either classic isotype conversions or non-classical isotype conversions. Classical isotype conversion occurs by recombination events involving one or more conversion sequence regions in a transgene. Non-classical isotype conversion can occur, for example, by homologous recombination between human σ _μ and human Σ _μ (δ-associated deletion). Among other things, separate non-classical conversion mechanisms such as recombination between transgenes and / or recombination between chromosomes may occur and isotype conversion may be performed.
[72] As used herein, the term "conversion sequence" refers to a DNA sequence responsible for converting recombination. A typical μ conversion region, “conversion donor” sequence, will be the 5 ′ (ie, up) of the construct region that is deleted during conversion recombination. The "converting recipient" region will be between the deleted construct region and the replacement constant region (eg, γ, ε, etc.). Since there is no specific site where recombination always occurs, typically the final gene sequence will not be predictable from the construct.
[73] As used herein, a "glycosylation pattern" is defined as a pattern of carbohydrate units covalently bound to a protein, more specifically an immunoglobulin protein. If one of ordinary skill in the art recognizes that the glycosylation pattern of the heterologous antibody is more similar to the glycosylation pattern in a transgenic animal species other than human than the pattern in the species from which the CH gene of the transgene is derived, Glycosylation patterns of heterologous antibodies can be characterized as being substantially similar to glycosylation patterns that occur naturally on antibodies produced by transgenic animal species other than humans.
[74] As used herein, the term “naturally occurring” as applied to a subject means that the subject can be found in its natural state. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and not deliberately modified by humans in the laboratory are naturally occurring sequences.
[75] As used herein, the term “rearranged” refers to the heavy or light chain immunoglobulin locus at the site immediately adjacent to the DJ or J fragment in the form where the V fragment encodes each of the essentially complete VH or VL domains. It means an array. The rearranged immunoglobulin locus can be identified by comparison with germline DNA, and the rearranged locus will have one or more recombinant tetrameric / 9mer homologues.
[76] As used herein, the terms "unrearranged" or "germline cell sequence" for a V segment refer to an arrangement immediately adjacent to a D or J segment by not recombining the V segment.
[77] As used herein, “nucleic acid molecule” includes DNA molecules and RNA molecules. The nucleic acid molecule may be single stranded or double stranded, but is preferably double stranded DNA.
[78] As used herein, the term “isolated nucleic acid molecule” for a nucleic acid encoding an antibody or antibody portion (eg, VH, VL, CDR3) that binds CD89 is within the nucleotide sequence encoding the antibody or antibody portion. There is no other nucleotide sequence encoding an antibody or antibody portion that binds to an antigen other than CD89, and the other sequence refers to a nucleic acid molecule that can naturally flank nucleic acids in human genomic DNA. In one embodiment, the human anti-CD89 antibody or portion thereof includes not only the nucleotide sequence or amino acid sequence of 14.1, 7.4, 8.2, but also shown in SEQ ID NOs: 1, 3, 5, 7, and 2, 4, 6, 8, respectively. Heavy chain (VH) and light chain (VL) variable regions with sequences are included.
[79] As disclosed and claimed herein, the sequences shown in SEQ ID NOS: 1-8 do not significantly affect the "conservative sequence variants", ie, the binding properties of an antibody encoded by said nucleotide sequence or an antibody having said amino acid sequence. Or variants of nucleotide and amino acid sequences that do not change. Such conservative sequence variants include those in which nucleotides and amino acids are substituted, added, and deleted. Variations can be introduced into SEQ ID NOS: 1-8 by standard techniques known in the art, such as site-directed mutations and PCR-mediated mutations. Conservative amino acid substitutions include replacing amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are defined in the art. These groups include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains Amino acids having (eg tyrosine, phenylalanine, tryptophan, histidine) are included. Thus, predicted non-essential amino acid residues in human anti-CD89 antibodies are preferably substituted with other amino acid residues in the same side chain group.
[80] Alternatively, in other embodiments, the mutations can be optionally introduced along the full length or partial sequence of the anti-CD89 antibody coding sequence by methods such as saturation mutations, and the resulting variant anti-CD89 antibodies are screened by binding activity. Can be.
[81] Thus, an antibody encoded by a nucleotide sequence (of the heavy and light chain variable regions) disclosed herein (ie, SEQ ID NOs: 1, 3, 5 and 7) and / or an amino acid sequence of the (heavy and light chain variable regions) disclosed herein ( That is, an antibody having SEQ ID NOs: 2, 4, 6 and 8) includes substantially similar antibodies encoded by or conservatively modified analogous sequences. Based on the partial (ie heavy and light chain variable region) sequences disclosed herein, such as SEQ ID NOS: 1-8, further discussion of methods for generating such substantially similar antibodies is given below.
[82] In the case of nucleic acids, the term “substantial homology” means that when two nucleic acids or their specific sequences are optimally aligned and compared, the nucleotides have the appropriate nucleotide insertion or deletion, and are at least about 80%, typically at least about 90-95%, More preferably about 98 to 99.5% or more identical. Alternatively, substantial homology exists when the fragment hybridizes with the complementary sequence of the strand under selective hybridization conditions.
[83] The percent identity between two sequences is a function of the number of identical sites divided by the sequences that need to be introduced for optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap (ie,% phase Homosexual = number of identical sites / total number of sites x 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm as described in the following non-limiting examples.
[84] The percent identity between two nucleotide sequences is determined by the GCG software package (http: /) using the NWSgapdna CMP matrix, and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. (available at www.gcg.com). The percent identity between two nucleotide or amino acid sequences was introduced into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. E. Meyers and W. Measurements can also be made using Miller's algorithm (Comput. Appl. Biosci., 4: 11-17 (1988)). In addition, the percent identity between two amino acid sequences refers to the Blossum 62 matrix or PAM250 matrix, and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 Needleman-Wunsch algorithm (J. Mol. Biol. (48): 444-453 introduced into the GAP program in the GCG software package (available at http://www.gcg.com)). 1970).
[85] The nucleic acid and protein sequences of the present invention can be used as "query sequences" to perform searches in public databases to identify, for example, related sequences. Such searches are described in Altschul, et al. (1990) J. Mol. Biol. 215: 403-10] and the NBLAST and XBLAST programs (version 2.0). A nucleotide sequence homologous to the nucleic acid molecule of the present invention can be obtained by performing a BLAST nucleotide search using the NBLAST program (Score = 100, word length = 12). A BLAST protein search can be performed using the XBLAST program (Score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain a gap alignment for comparison purposes, see Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402, Gapped BLAST can be used. When using the BLAST and Gapped BLAST programs, the initial parameters of each program (eg XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov).
[86] The nucleic acid may be present in the complete cell or cell lysate in partially purified or substantially pure form. Nucleic acids can be prepared by other techniques such as alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art for other cellular components or other contaminants (eg, When isolated and purified from cellular nucleic acid or protein, the nucleic acid is "isolated" or "in substantially pure form" (F. Ausubel, et al., Ed. Current Protocols in Molecular Biology , Greene Publishing and Wiley Interscience, New York (1987).
[87] Nucleic acid compositions of the invention from cDNAs, genomes, or mixtures thereof are often present in native sequences (except for modified restriction sites, etc.), but can be mutated according to standard techniques to provide gene sequences. In the case of coding sequences, these mutations can affect the desired amino acid sequence. In particular, DNA sequences that are substantially homologous to or derived from native V sequences, D sequences, J sequences, constant sequences, conversion sequences, and other such sequences described herein are also contemplated (where “derived” means Meaning that the sequence is identical to or modified from another sequence).
[88] When a nucleic acid is positioned to have a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if a promoter or enhancer affects the transcription of the sequence, it is operably linked to the coding sequence. In the case of transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous and, where necessary, two protein coding regions are contiguously linked in a reading frame. In the case of a conversion sequence, operably linked means that the sequence can perform conversion recombination.
[89] As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering other linked nucleic acids. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors can autonomously replicate in host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with bacterial replication origin). Other vectors (eg, non-episomal mammalian vectors) can also be inserted into the genome of the host cell upon introduction into the host cell and replicate with the host genome. Moreover, some vectors can express genes operably linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably since the plasmid is in the form of the most commonly used vector. However, the present invention includes other forms of expression vectors with the same function as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).
[90] As used herein, the term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It is to be understood that these terms mean not only the particular subject cell but also the progeny of such cell. Because some mutations may occur in subsequent cells due to mutations or environmental effects, in fact these progeny may not be identical to the parent cell but are still included within the scope of the term "host cell" as used herein. . Recombinant host cells include, for example, CHO cells and lymphocytic cells.
[91] Various aspects of the invention are described in more detail in the following paragraphs.
[92] I. Production of Human Antibodies Against CD89
[93] Monoclonal antibodies (mAb) of the invention can be produced by a variety of techniques including conventional monoclonal antibody methodologies, such as standard somatic hybridization techniques (Kohler and Milstein, Nature 256: 495 (1975)). While somatic hybridization methods are generally preferred, other techniques for producing monoclonal antibodies (eg viral or neoplastic transformation of B lymphocytes) may be used.
[94] Preferred animal systems for making hybridomas are murine systems. Hybridoma production in mice is a very well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion methods are also known.
[95] In a preferred embodiment, human monoclonal antibodies against CD89 can be generated using transgenic mice carrying parts of the human immune system rather than mouse systems. These transgenic mice, referred to herein as “HuMAb” mice, encode unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with target mutations that inactivate endogenous μ and κ chain loci. Human immunoglobulin gene mini locus (Lonberg, N. et al. (1994) Nature 368 (6474): 856-859). Thus, the mice show reduced expression of IgM or κ in mice, and the introduced human heavy and light chain transgenes produce high-affinity human IgGκ monoclonal antibodies through class switching and somatic mutation in response to immunization (Lonberg , N. et al. (1994), reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci 764: 536-546). The preparation of HuMab mice is described in paragraph II and in Taylor, L. et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90: 3720-3724; Choi et al., (1993) Nature Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 2912-2920; Lonberg et al., (1994) Nature 368 (6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764: 536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. International); U.S. Patent 5,545,807 to Surani et al .; WO 98/24884 published June 11, 1998; WO 94/25585, published November 10, 1994; WO 93/1227, published June 24, 1993; WO 92/22645 published December 23, 1992; See also WO 92/03918, published March 19, 1992, the disclosures of which are hereby incorporated by reference in their entirety. Alternatively, human anti-CD89 antibodies can be produced using the HC012 transgenic mice described in Example 2.
[96] HuMab immunization
[97] To produce fully human monoclonal antibodies against CD89, Lonberg, N. et al. (1994) Nature 368 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884, HuMab mice can be immunized with purified CD89 antigen preparations or preparations rich in these antigens, and / or cells expressing CD89. Preferably, mice are 6-16 weeks old at the first infusion. For example, HuMab mice can be immunized by intraperitoneal injection of an isolated CD89 antigen preparation or a preparation rich in this antigen (eg, purified from LNCaP cells expressing CD89) (5-20 μg). If antibodies are not generated by immunization with a purified CD89 antigen preparation or a preparation rich in this antigen, mice may be immunized with cells expressing CD89 (eg, tumor cell lines) to promote an immune response.
[98] Through accumulated experience with various antigens, HuMAb transgenic mice have been initially immunized with intraperitoneal injection (IP) of antigens from complete Freund's adjuvant, followed by Freund's incomplete immunoadjuvant (incomplete). It has been found that it responds best to weekly IP immunization (up to 6 total) with antigens in Freund's adjuvant. The immune response can be observed over the course of the immunization protocol using plasma samples obtained by retroorbital bleeding. Plasma can be screened by ELISA (described below) and mice with sufficient titers of anti-CD89 human immunoglobulin can be used for fusion. Three days before the spleen is removed at the expense of the mouse, the antigen can be injected intravenously and boosted. It may be necessary to perform two to three fusions for each antigen. Several mice will be immunized for each antigen. For example, a total of 12 HuMAb mice of the HC07 and HC012 strains can be immunized.
[99] Hybridoma Production Producing Human Monoclonal Antibodies Against CD89
[100] Based on standard protocols, mouse splenocytes can be isolated and fused with mouse myeloma cell lines with PEG. The prepared hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice using 50% PEG are fused with a number of cells corresponding to one sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580). Let's do it. About 2 × 10 ⁵ cells were plated on flat bottom microtiter plates, followed by 20% Fetal Clone Serum, 18% “653” Control Medium, 5% Origen (IGEN), 4 mM L- Glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin and 1 x HAT Incubate for two weeks in selection medium containing (Sigma; HAT added 24 hours after fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Each well was then screened by ELISA for human anti-CD89 monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is usually observed after 10-14 days. Hybridomas secreting antibodies can be replated, screened again, and subclones can be screened two or more times by limiting dilution if still positive for human IgG, anti-CD89 monoclonal antibodies. Subsequently, stable subclones are cultured in vitro to produce a small amount of antibody in the tissue culture medium for characterization.
[101] Use of Partial Antibody Sequences to Express Complete Antibodies
[102] Antibodies interact with target antigens primarily through amino acid residues located within six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences in the CDRs are more diverse than the sequences outside the CDRs between the respective antibodies. Because CDR sequences are involved in most antibody-antigen interactions, by producing expression vectors comprising CDR sequences from naturally occurring specific antibodies, implanted on the framework sequences of other antibodies with different properties, It is possible to express recombinant antibodies that mimic the characteristics of certain antibodies (Riechmann, L. et al., 1988, Nature 332: 323-327; Jones, P. et al., 1986, Nature 321: 522-525). Queen, C. et al., 1989, Proc. Natl. Acad. Sci. USA 86: 10029-10033. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. The germline sequence will differ from the mature antibody gene sequence because the germline sequence does not contain a fully assembled variable region formed by V (D) J ligation upon B cell maturation. In addition, the germline gene sequence will also differ from each nucleotide sequence of a high affinity second repertoire antibody, each evenly distributed in the variable region. For example, somatic mutations occur at a relatively low frequency in the amino-terminal portion of the framework region. For example, somatic mutations occur at relatively low frequencies in the amino terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4. Moreover, many somatic mutations do not significantly change the binding properties of the antibody. For this reason, it is not necessary to obtain the full DNA sequence of a particular antibody to reproduce a complete recombinant antibody with binding properties similar to the original antibody (filed March 12, 1999, which is incorporated herein by reference through this nomenclature). PCT / US99 / 05535). Partial heavy and light chain sequences lying in the CDR regions are typically sufficient for this purpose. The partial sequence is used to determine germline variable and linkage gene segments that contribute to recombinant antibody variable genes. The germline sequence is then used to fill in the deletion portion of the variable region. Heavy and light chain leader sequences are cleaved upon protein maturation and do not contribute to the properties of the final antibody. For this reason, there is a need to use the corresponding germline leader sequences for expression constructs. To add a deletion sequence, the cloned cDNA sequence can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire synthetic variable region clones can be made by synthesizing the entire variable region into a set of short overlapping oligonucleotides and binding by PCR amplification. This method has certain advantages, such as elimination or inclusion of certain restriction sites or optimization of certain codons.
[103] The nucleotide sequences of the heavy and light chain transcripts from the hybridomas were used to design overlapping sets of synthetic oligonucleotides to produce synthetic V sequences with the same amino acid coding ability as the native sequence. The sequences of the synthetic heavy and kappa chains are interleaved in three ways (repeated nucleotide sequences to facilitate the synthesis and PCR amplification of oligonucleotides; Kozak's rule (Kozak, 1991, J. Biol. Chem) 266: 19867-19870), and the optimal translation initiation site is incorporated; and the HindIII site is introduced above the translation initiation site).
[104] In the heavy and light chain variable regions, the optimized coding strand sequence and the corresponding non-coding strand sequence are cleaved into approximately 30 to 50 nucleotides near approximately the center of the non-coding oligonucleotide. Thus, in each chain the oligonucleotides can be assembled into overlapping double stranded sets spanning 150 to 400 nucleotide fragments. Thereafter, the pool is used as a template to prepare a PCR amplification product of 150 to 400 nucleotides. Typically, a single set of variable region oligonucleotides will be divided into two pools, which are amplified separately to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to create complete variable regions. It may also be desirable to include overlapping fragments of heavy or light chain constant regions (including the BbsI region of the kappa light chain or the AgeI region of the gamma heavy chain) upon PCR amplification to make fragments that can be easily cloned into the expression vector construct. .
[105] The reconstituted heavy and light chain variable regions are then combined with the cloned promoter sequence, translation initiation sequence, constant region sequence, 3 ′ untranslated sequence, polyadenylation sequence and transcription termination sequence to form an expression vector construct. Heavy and light chain expression constructs can be combined in a single vector, transfected simultaneously into host cells, sequentially transfected, or individually transfected into each host cell and then fused to form host cells expressing the two chains. can do.
[106] The plasmids used in the construction of expression vectors for human IgGκ are described below. This plasmid can be used to reconstruct the heavy and light chain minigenes with the V heavy and V kappa light chain cDNA sequences amplified by PCR. These plasmids can also be used to express fully human antibodies, chimeric IgG1κ antibodies or IgG4κ antibodies. Similar plasmids can be prepared for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.
[107] Thus, in another aspect of the invention, the structural features of the human anti-CD89 antibodies 14.1, 7.4 or 8.2 of the invention are structurally related humans that retain one or more functional properties of the antibodies of the invention such as bind to CD89. It is used to produce anti-CD89 antibodies. More specifically, one or more CDR regions of 14.1, 7.2 or 8.2 can be recombinantly combined with known human framework regions and CDRs to produce further recombinantly engineered human anti-CD89 antibodies of the invention. .
[108] Thus, in another embodiment the invention provides an amino acid sequence (or sequence) selected from (1) a human heavy chain framework region and a human heavy chain CDR, wherein at least one human heavy chain CDR is selected from the amino acid sequence of the CDRs shown in FIG. 2 or the corresponding amino acid residue of SEQ ID NO: 6); And (2) the human light chain framework region and human light chain CDRs, wherein the one or more human light chain CDRs are selected from the amino acid sequences of the CDRs shown in FIG. 2 or 4 (or the corresponding amino acid residues of SEQ ID NO: 4 or SEQ ID NO: 8). A method of making an anti-CD89 antibody, comprising preparing an antibody, wherein the antibody has the ability to bind CD89. The ability of the antibody to bind CD89 can be measured using standard binding assays such as those described in the Examples (eg, ELISA).
[109] Since the antibody heavy and light chain CDR3 domains are well known in the art to play a particularly important role in the binding specificity / affinity of the antibody for antigen, the recombinant antibodies of the invention prepared as described above are preferably 14.1 Heavy chain and light chain CDR3 of 7.4 or 8.2. The antibody may further comprise a CDR2 of 14.1, 7.4 or 8.2. Accordingly, the present invention is directed to (1) human heavy chain framework region, human heavy chain CDR1 region, human heavy chain CDR2 region and human heavy chain CDR3 region, wherein the human heavy chain CDR3 region is CDR3 of 14.1, 7.2 and 8.2 shown in FIG. (Or the corresponding amino acid residue of SEQ ID NO: 2 or SEQ ID NO: 6); And (2) human light chain framework regions, human light chain CDR1 regions, human light chain CDR2 regions and human light chain CDR3 regions, wherein the human light chain CDR3 regions are CDR3 of 14.1, 7.4 and 8.2 shown in FIGS. 2 and 4 (or SEQ ID NO: 4 or Is selected from the corresponding amino acid residues of SEQ ID NO: 8), wherein the antibody binds to CD89. The antibody may further comprise heavy chain CDR2 and / or light chain CDR2 of 14.1, 7.4 or 8.2. The antibody may further comprise heavy chain CDR1 and / or light chain CDR1 of 14.1, 7.4 or 8.2.
[110] Preferably, CDR1, CDR2 and / or CDR3 of the engineered antibody comprise an amino acid sequence identical to the amino acid sequence of 14.1, 7.4 or 8.2 disclosed herein. However, one of ordinary skill in the art will understand that while still retaining the ability of an antibody to bind CD89 effectively, it may be possible to slightly deviate from the correct CDR sequences of 14.1, 7.4 and 8.2 (eg, conservative substitutions). Thus, in other embodiments, the engineered antibody may be composed of one or more CDRs that are 90%, 95%, 98% or 99.5% identical, for example, to one or more CDRs of 14.1, 7.4 or 8.2.
[111] In addition to simply binding to CD89, engineered antibodies such as those described above
[112] 1) binds to living cells expressing CD89;
[113] 2) high binding affinity for CD89;
[114] 3) binding to specific epitopes on CD89 (to eliminate the possibility of competing for binding to the same epitope when used with monoclonal antibodies with complement activity);
[115] 4) opsonization of CD89 expressing cells; And / or
[116] 5) mediate growth inhibition, phagocytosis and / or killing of CD89 expressing cells in the presence of human effector cells.
[117] And for retaining other functional properties of the antibodies of the invention.
[118] Characterization of Human Monoclonal Antibody Binding to CD89
[119] To characterize the binding of the human monoclonal CD89 antibodies of the invention, the serum of immunized mice can be tested, for example by ELISA. In a typical (but non-limiting) example of the ELISA protocol, the microtiter plate is coated with 0.25 μg / ml of purified CD89 in PBS and then blocked with 5% bovine serum albumin in PBS. Plasma dilutions of CD89-immunized mice are added to each well and incubated at 37 ° C. for 1-2 hours. The plates are washed with PBS / Tween and then incubated at 37 ° C. for 1 hour with the addition of goat-anti-human IgG Fc-specific polyclonal reagent conjugated to alkaline phosphatase. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at OD of 405-650. Preferably, mice with the highest titers will be used for fusion.
[120] Such ELISA assays can also be used to screen hybridomas that are positively responsive to the CD89 immunogen. Hybridomas that bind with high binding to CD89 will be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells (analyzed by ELISA) can be selected to prepare 5-10 vial cell banks (stored at -140 ° C) and to purify the antibody. .
[121] To purify human anti-CD89 antibodies, selected hybridomas can be cultured in a 2-liter spinner-flask to purify monoclonal antibodies. The supernatant can be filtered and concentrated and then subjected to affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be analyzed by gel electrophoresis and high performance liquid chromatography to confirm purity. The buffer solution can be replaced with PBS and the concentration can be measured by OD ₂₈₀ using 1.43 extinction coefficient. Monoclonal antibodies can be aliquoted and stored at -80 ° C.
[122] To determine the binding of selected human anti-CD89 monoclonal antibodies to specific epitopes, commercially available reagents (Pierce, Rockford, IL) can be used to biotinylate each antibody. As described above, CDISA-coated ELISA plates can be used to conduct competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies. Binding of biotinylated mAbs can be detected with strep-avidin alkaline phosphatase probes.
[123] To determine the isotype of purified antibodies, isotype ELISAs can be performed. For example, wells of microtiter plates can be coated with 10 μg / ml of anti-human Ig overnight at 4 ° C. After blocking with 5% BSA, the plate is reacted with 10 μg / ml monoclonal antibody or purified isotype control at room temperature for 2 hours. The wells can then be reacted with probes conjugated with human IgG1 or human IgM-specific alkaline phosphatase. The plate is developed and analyzed as described above.
[124] Flow cytometry can be used to demonstrate that monoclonal antibodies bind to living cells expressing CD89. Illustrating a typical (but non-limiting) flow cytometer protocol, cell lines expressing CD89 (grown under standard growth conditions) were monoclonal at varying concentrations in PBS containing 0.1% Tween 80 and 20% mouse serum. Mix with antibody and incubate at 37 ° C. for 1 hour. After washing, the cells are reacted with anti-human IgG antibodies labeled with Fluorescein under the same conditions as primary antibody staining. Samples can be analyzed with FACScan instruments using light, and lateral scattering on single cells. Other assays using fluorescence microscopy may be used in conjunction with or instead of flow cytometer assays. Cells can be stained and examined by fluorescence microscopy as described above. Individual cells can be seen in this way, but sensitivity may be reduced depending on the density of the antigen.
[125] Western blotting can further test for reactivity of the anti-CD89 human IgG with the CD89 antigen. For example, cell extracts from cells expressing CD89 can be prepared and analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the isolated antigens are transferred to nitrocellulose membranes and blocked with 20% mouse serum and detected with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate purification (Sigma Chem. Co., St. Louis, Mo.).
[126] II. Preparation of non-human transgenic animals producing human monoclonal anti-CD89 antibodies
[127] In another aspect, the invention preferably provides a non-human transgenic animal (eg, a transgenic mouse) that has a high affinity and is capable of expressing a human monoclonal antibody that specifically binds to CD89. to provide. In a preferred embodiment, said non-human transgenic animal (eg, transgenic mouse (HuMab mouse)) has a genome comprising a human heavy chain transgene and a light chain transgene. In one embodiment, the non-human transgenic animal (eg, transgenic mouse) is immunized with a purified CD89 antigen or an agent rich in CD89 antigen, and / or cells expressing CD89. Preferably, said non-human transgenic animals (eg, transgenic mice), through VDJ recombination and isotype conversion, are capable of multiple isotypes of human monoclonal antibodies against CD89 (eg, IgG, IgA and / or IgE) can be produced. Isotype conversion can occur, for example, by classical or non-classical isotype conversion.
[128] Designing a non-human transgenic animal that responds to foreign antigen stimulation using a heterologous antibody repertoire requires that the heterologous immunoglobulin transgene is contained within the transgenic animal and works correctly through the B-cell developmental pathway. . In a preferred embodiment, the correct operation of the heterologous heavy chain transgene includes isotype conversion. Thus, the transgenes of the present invention are isotype converted, and (1) high levels of cell-type specific expression, (2) functional gene rearrangement, (3) activation of allele exclusion and response to allele exclusion, And (4) sufficient expression of the main repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response.
[129] It is not necessary to meet all of the above criteria. For example, in embodiments where the function of the endogenous immunoglobulin locus is impaired in transgenic animals, the transgene does not need to activate allele exclusion. In addition, for embodiments wherein the transgene comprises a functionally rearranged heavy and / or light chain immunoglobulin gene, a second criterion of functional gene rearrangement is not necessary, at least if the transgene has already been rearranged. For background art of molecular immunology, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N. Y.].
[130] In some embodiments, transgenic animals other than humans used to produce human monoclonal antibodies of the invention are heterologous immunoglobulin heavy and light chain transgenes, non-rearranged heterologous rearranged in the germline of a transgenic animal. Immunoglobulin heavy and light chain transgenes, or combinations thereof. Heavy chain transgenes each contain one or more C _H genes. In addition, heavy chain transgenes may have functional isotype conversion sequences that may support isotype conversion of heterologous transgenes that encode multiple C _H genes in B-cells of a transgenic animal. This conversion sequence occurs naturally in the germline immunoglobulin locus from the species serving as the source of the transgene C _H gene, or in the species (transgenic animal) in which this conversion sequence will receive the transgene construct. Can be derived from those that occur. For example, human transgene constructs used to prepare transgenic mice have a higher frequency of isotype conversion events when this construct contains a conversion sequence similar to that naturally occurring at the mouse heavy chain locus. This may be as if the mouse switch sequence optimizes operation using the mouse switch recombinase enzyme system but not for the human switch sequence. Conversion sequences can be isolated and cloned by conventional cloning methods, or synthesized by de novo by overlapping synthetic oligonucleotides designed based on public sequence information related to immunoglobulin conversion region sequences. (Mills et al., Nucl.Acids Res. 15: 7305-7316 (1991); Sideras et al., Intl. Immunol. 1: 631-642 (1989), incorporated herein by this reference. ] Reference).
[131] In each of these transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are present in significant proportions (10% or more) in the B-cells of the transgenic animal.
[132] The transgene used to prepare the transgenic animal of the invention encodes one or more variable gene segments, one diversity gene segment, one joining gene segment and one or more constant region gene segments. Heavy chain transgenes containing DNA are included. Immunoglobulin light chain transgenes include DNA encoding one or more variable gene segments, one linking gene segment and one or more constant region gene segments. Gene segments encoding heavy and light chain gene segments are derived from or correspond to DNA encoding immunoglobulin heavy and light chain gene segments from species not comprised of non-human transgenic animals. Heterogeneous for genic animals. In one aspect of the invention, the transgene is configured such that each gene segment is not rearranged, that is, not rearranged to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes maintain recombination (functional rearrangement) of the V, D, and J gene segments, and preferably all or part of the D region gene segments, when exposed to the CD89 antigen, are transgenic other than human. The incorporation into rearranged immunoglobulin heavy chains generated in the genic animal is maintained.
[133] In a separate embodiment, the transgene comprises a "minisite" that is not rearranged. Such transgenes typically comprise a substantial portion of a subset of V gene segments as well as C, D and J segments. In such transgene constructs, a variety of regulatory sequences, such as promoter sequences, enhancer sequences, class conversion region sequences, splice-donor and splice-receptor sequences for RNA processing, and recombinant signal sequences, etc., can be derived from heterologous DNA. The corresponding sequence derived. Such regulatory sequences may be incorporated into transgenes from the same species or related species as animals other than humans used in the present invention. For example, human immunoglobulin gene segments can bind to transgenes having rodent immunoglobulin enhancer sequences used in transgenic mice. Alternatively, synthetic regulatory sequences can be incorporated into the transgene, where the synthetic regulatory sequences are not homologous to functional DNA sequences known to occur naturally in the mammalian genome. Synthetic regulatory sequences are designed according to common rules such as, for example, specifying acceptable sequences of splice-receptor sites or promoter / enhancer motifs. For example, the mini locus is internal to the non-essential DNA portion (eg, intervening sequence; intron or portion thereof) relative to the naturally occurring germ line Ig locus (ie, not at the end of that portion). A portion of a genomic immunoglobulin locus having one or more deletions.
[134] In a preferred embodiment of the invention, the transgenic animal used to produce a human antibody against CD89 is a transgene (eg, described in Example 12 of WO 98/24884, the contents of which are incorporated herein by reference). light chain trans described in Examples 5, 6, 8 or 14 of WO 98/24884, comprising at least 1 copy, typically 2 to 10 copies, sometimes 25 to 50 copies or more. The animal is crossed with an animal comprising a single copy of the gene, and its offspring are crossed with the J _H deleted animal described in Example 10 of WO 98/24884. Animals are bred to produce homozygotes for each of these three properties. These animals have the following genotypes: A single copy of the unrearranged human heavy chain mini locus (per chromosome haploid set) (described in Example 12 of WO 98/24884); A single copy of a rearranged human κ light chain construct (per chromosome haploid set) (described in Example 14 of WO 98/24884); And deletions in each of the endogenous mouse heavy chain loci, in which all of the functional J _H segments are removed (described in Example 10 of WO 98/24884). These animals were crossed with mice homozygous for the deletion of the J _H fragment (Example 10 of WO 98/24884) to produce progeny homozygous for the J _H deletion and semizygous for the human heavy and light chain constructs. do. Antigens are injected into the produced animals and these animals are used to produce human monoclonal antibodies against the antigens.
[135] B cells isolated from these animals are monospecific for human heavy and light chains because they contain only a single copy of each gene. In addition, the B cells are human or mouse heavy chains because both endogenous mouse heavy chain gene copies are not functional due to deletion across the J _H region introduced as described in Examples 9 and 12 of WO 98/24884. Will be monospecific for. In addition, substantial portions of B cells are characterized by the expression of a single copy of the rearranged human κ light chain genes, alleles and isotypes to exclude rearrangements of endogenous mouse κ and lambda chain genes in a substantial portion of the B cells. It will be monospecific for human or mouse light chains.
[136] Transgenic mice of the preferred embodiments will produce immunoglobulins comprising significant repertoires, ideally substantially similar to immunoglobulins of natural mice. Thus, for example, in embodiments where the endogenous Ig gene is inactivated, the total level of immunoglobulin is about 0.1 to 10 mg per ml of serum, preferably 0.5 to 5 mg per ml of serum, ideally At least about 1.0 mg per ml of serum. When transgenes are introduced into transgenic mice capable of performing the conversion from IgM to IgG, the ratio of IgG to IgM in the serum of adult mice is preferably about 10: 1. The IgG to IgM ratio will be much lower in immature mice. In general, more than about 10%, preferably 40-80%, of the spleen and lymph node B cells express human IgG protein exclusively.
[137] The repertoire is ideally similar to that seen in non-transgenic mice and will generally be at least about 10% thereof, and preferably at least 25-50%. In general, at least about 1,000, preferably at least 10 ⁴ to 10 ⁶ different immunoglobulins (ideally IgG) are produced mainly depending on the number of different V, J and D regions introduced into the mouse genome. Will be. Such immunoglobulins will typically recognize at least about one half of the highly antigenic proteins (eg, staphylococcus protein A). Typically, the immunoglobulin is at least about 10 ⁷ M ⁻¹ , preferably at least about 10 ⁹ M ⁻¹ , more preferably about 10 ¹⁰ M ⁻¹ , 10 ¹¹ M ⁻¹ , 10 ¹² with respect to the preselected antigen An affinity of M ⁻¹ or higher (eg, 10 ¹³ M ⁻¹ or lower).
[138] In some embodiments, it may be desirable to produce mice with predetermined repertoires to limit the selection of V genes that are present in antibody responses against predetermined antigen types. Heavy chain transgenes having a predetermined repertoire can include human VH genes that are primarily used in antibody responses to predetermined antigenic types in humans, for example. Alternatively, some VH genes are less likely to encode a high affinity V region for a given antigen for a variety of reasons (e.g., less likely to carry out somatic mutations and affinity enhancement; or in some humans). May be excluded from the defined repertoire). Thus, prior to rearrangement of transgenes with various heavy or light chain gene segments, such gene segments can be readily identified as gene segments from organism species other than transgenic animals, for example by hybridization or DNA sequencing.
[139] As described above, the transgenic mice of the present invention can be immunized with purified CD89 antigen preparations or preparations rich in CD89 antigens and / or CD89 expressing cells. This mouse will perform class-conversion via transfection recombination (cis-conversion) in the transgene to produce B cells expressing immunoglobulins reactive to CD89. The immunoglobulins may be human sequence antibodies, wherein the heavy and light chain polypeptides thereof are encoded by human transgene sequences, which may include germline-coding sequences as well as sequences induced by somatic mutations and V region recombinant junctions. Such human immunoglobulin sequences are encoded by human V _L or V _H gene segments, and human J _L or J _L segments even when other non-germline sequence sequences are present as a result of somatic mutations and specific VJ and VDJ recombination linkages. May be referred to as substantially identical to the resulting polypeptide sequence. For such human sequence antibodies, the variable regions of each chain are typically encoded at least 80% by human germline V, J and D gene segments (in the case of heavy chains), often at least 85% of the variable regions on the transgene Encoded by the human germline sequence present in, often at least 90 or 95% of the variable region sequences are encoded by the human germline sequence present on the transgene. However, since non-germline sequences are introduced by somatic mutations and VJ and VDJ ligation, human sequence antibodies are often not encoded by human V, D or J gene segments found in human transgenes in mouse germline lines. Some variable region sequences (less frequently more often constant region sequences). Typically, such non-germline cell sequences (or respective nucleotide positions) will be dense in or near the CDRs or in regions known to be densely populated with somatic mutations.
[140] Human sequence antibodies that bind to a predetermined antigen are isotype converted such that human antibodies comprising human sequence γ chains (eg γ1, γ2a, γ2B or γ3) and human sequence light chains (eg κ) are produced Can be formed from. Such isotype-converted human sequence antibodies are often as a result of affinity maturation and selection of B cells by the antigen, especially as a result of subsequent (or subsequent) antigen administration, typically within the variable region. Often with about 10 residues or one or more somatic mutations in the CDRs. Such high affinity human sequence antibodies are at least 1 × 10 ⁹ M ⁻¹ , typically at least 5 × 10 ⁹ M ⁻¹ , often at least 1 × 10 ¹⁰ M ⁻¹ , sometimes 5 × 10 ¹⁰ M ⁻¹ to 1 × It may have a binding affinity of 10 ¹¹ M ⁻¹ or more.
[141] Another aspect of the invention relates to a mouse derived from a mouse that can be used to produce hybridomas expressing human monoclonal antibodies that bind to CD89 with high affinity (eg greater than 2 × 10 ⁹ M ⁻¹ ). Relates to B cells. Thus, in another embodiment of the invention, such hybridomas are used to produce a composition comprising an immunoglobulin that binds CD89 with an affinity constant (K _a ) of at least 2 × 10 ⁹ M ⁻¹ , wherein the Immunoglobulins,
[142] (1) a light chain variable region having a polypeptide sequence substantially identical to the polypeptide sequence encoded by the human V _L gene segment and the human J _L segment, and (2) a polypeptide sequence substantially identical to the polypeptide sequence encoded by the human C _L gene segment Human sequence light chains consisting of a light chain constant region having a polypeptide sequence; And
[143] (1) a heavy chain variable region having a polypeptide sequence substantially identical to a polypeptide sequence encoded by a human V _H gene segment, optionally a D region and a human J _H segment, and (2) a polypeptide encoded by a human C _H gene segment Human sequence heavy chain consisting of a constant region having a polypeptide sequence substantially identical to a sequence
[144] It includes.
[145] The development of high affinity human monoclonal antibodies against CD89 is facilitated by a method of extending the repertoire of human variable region gene segments in transgenic mice with a genome comprising an integrated human immunoglobulin transgene. Introducing into the genome a V gene transgene comprising a V region gene segment that is not present in the integrated human immunoglobulin transgene. Frequently, the V region transgenes are naturally occurring in the human genome or can be spliced to each other by recombinant methods, and the yeast artificial chromosomes comprising part of the human V _H or V _L ( _Vκ ) gene segment analysis. And V gene segments may be included out of sequence or omitted. Frequently, at least five functional V gene segments are contained on the YAC. In a variation of this method, a transgenic mouse produced by the V repertoire expansion method can be made, which mouse is made by a V region gene segment present on the V region transgene, and a C region encoded in the human Ig transgene. Express an immunoglobulin chain comprising the variable region sequence that is encoded. By the V repertoire extension method, transgenic mice having five or more distinct V genes can be produced, and mice with about 24 or more V genes can also be produced. Some V gene segments may be non-functional (eg, pseudogenes, etc.), and such fragments may be retained or optionally deleted by recombinant methods known to those skilled in the art, if necessary.
[146] If a mouse germline is engineered to include a functional YAC with an extended V segment repertoire that is substantially absent from a human Ig transgene with J and C gene segments, the functional YAC with an extended V segment repertoire may be replaced by another human Ig. It can be delivered and introduced into other genetic backgrounds, including the background when introduced into a mouse germ cell line with a transgene. Multifunctional YACs with an extended V segment repertoire can be introduced into the germ cell line to work with human Ig transgenes (or multiple human Ig transgenes). Although referred to herein as a YAC transgene, when the transgene is integrated into the genome, the transgene may substantially lack yeast sequences, such as those required for spontaneous replication in yeast, such sequences being yeast Genetic manipulation (eg, restriction truncation and pulse-field gel electrophoresis or other suitable method) after replication in E. coli is no longer needed (ie, prior to introduction into mouse ES cells or mouse prozygote) May be optionally removed). Methods of enhancing the expression properties of human sequence immunoglobulins include raising transgenic mice with human Ig transgenes, and optionally transgenic mice with YACs with an extended V segment repertoire. Both V _H and V _L gene segments may be present on YAC. Such transgenic mice can be introduced to any background required by one of skill in the art, including backgrounds with human Ig transgenes and / or other transgenes in humans, including transgenes encoding human lymphocyte proteins. The present invention also provides high affinity human sequence immunoglobulins produced by transgenic mice having an extended V region repertoire YAC transgene. Although the foregoing describes the preferred embodiments of the transgenic animals of the present invention, other embodiments, which fall into the following four categories, are also contemplated by the present invention.
[147] I. Transgenic animals with unrearranged heavy and rearranged light chain immunoglobulin transgenes;
[148] II. Transgenic animals with unrearranged heavy and light chain immunoglobulin transgenes;
[149] III. Transgenic animals with rearranged heavy and unrearranged light chain immunoglobulin transgenes; And
[150] IV. Transgenic animals with rearranged heavy and light chain immunoglobulin transgenes.
[151] Among these transgenic animal categories, the preferred sequence is II> I> III> IV, where the endogenous light chain gene (or at least κ gene) is knocked out by homologous recombination (or other method) and I> II> III> IV, where the endogenous light chain gene is not knocked out and must be dominant by allelic exclusion.
[152] III. Bispecific / multispecific molecules that bind to CD89
[153] In another embodiment of the present invention, a human monoclonal antibody or antigen binding portion thereof against CD89 is derivatized or linked to another functional molecule, such as another peptide or protein (eg Fab 'fragment), Bispecific or multispecific molecules can be generated that bind to multiple binding sites or target epitopes. For example, an antibody or antigen binding moiety of the invention can be functionally linked to one or more other binding molecules, such as other antibodies, antibody fragments, peptides or binding mimetics, e.g. chemical coupling, gene fusion or non-covalent Linking, etc.). In addition, by linking the antibody or antibody fragment to an antigen such as a tumor antigen or the like, the antigen is targeted to immune cells expressing CD89, for example to enhance the process by which the antigen is internalized and then presented, ultimately stimulating an immune response. You can do that.
[154] Thus, the present invention includes bispecific and multispecific molecules that include one or more first binding specificities for CD89 and a second binding specificity for a target epitope of a target cell, such as a tumor cell. Bispecific antibodies, including murine antibodies against CD89 and second antibodies against tumor antigens, have proven to be effective anti-tumor agents. See, eg, Valerius, T. et al. (1997) Blood 90: 4485-4492 (anti-antibody of FcαRI x HER-2 / Neu bispecific antibodies used with or without G-CSF therapy). Tumor activity), Elsasser, D. et al. (1999) Anticancer Res. 19: 1525-1528 (FcαRI x EGFR bispecific antibodies used with or without G-CSF or GM-CSF therapy) Describes the anti-tumor activity of), [Stockmeyer, B. et al. (2000) J. Immunol. 165: 5954-5961] (FcαRI x CD20 used with or without G-CSF or GM-CSF therapy) Describes the anti-tumor activity of bispecific antibodies), [Stockmeyer, B. et al. (2001) J. Immunol. Methods 248: 103-111], with or without G-CSF or GM-CSF therapy Anti-tumor activity of FcαRI x CD20 and FcαRI x HER-2 / neu bispecific antibodies used without) and Sundarapandiyan, K. et al. (2001) J. Immunol. Methods 248: 113-123 The anti-tumor activity of FcαRI x CD30 bispecific antibodies See also)). Human anti-CD89 antibodies of the invention can be used in the same type of bispecific constructs as anti-tumor agents.
[155] Tumor cells that may be targeted include tumor cells of any cancer type, including breast, ovary, prostate, testes, lungs, colon, rectum, pancreas, liver, central nervous system, kidney, head, neck, bone, blood, and lymphatic system cancers. to be. Preferred target antigens include carcinoblastic antigen (CEA), gastrin releasing peptide receptor antigen, mucin antigen, EGF-R, HER2 / neu , HER3, HER4, CD20, CD30, MAGE antigen, SART antigen, MUC1 antigen, c-erb- 2 antigens and TAG 72 are included. TAG 72 is found, for example, in breast, colon and ovarian tumors. Antigen binding regions specific for CEA can be, for example, regions from single-chain antibodies called MFE-23, see Casey et al. (1994) J. Immunol. Methods 179: 105 and Chester et al. (1994) Lancet 343: 455). Anti-HER2 / neu antibodies are produced by cell line 520C9 [Ring et al. 1991 J. Immunol. 28: 915-917. Antibody H425 is a humanized form of anti-EGF-R antibody M425. Antibodies to TAG 72 include PCT Application Publication WO 93/11161, PCT Application Publication WO 90/04410 (corresponding to licensed European Patent No. 365 997), PCT Application Publication WO 93/12231 and PCT Application Publication WO 89/01783 Monoclonal antibody cc49 as described in Other antigens can be antigens associated with B cell lymphomas such as HLA-DR, CD74, CD79, CD20, CD30, CD37 and CD19. Antigens associated with other blood diseases are also within the scope of the present invention. Accordingly, the present invention also provides a method of treating blood cell disorders such as leukemia and lymphoma.
[156] Breast and ovarian cancers can be sex hormone-dependent cancers. Breast tumors may be characterized by abnormally expressed receptors such as human-EGF-like receptor family (HER), such as receptors of HER-2, -3 and -4. The invention is not limited to this embodiment of the HER antigen. Heregulin, a natural HER ligand, is a human monoclonal antibody such as bispecific antibody (BsAb) or multispecific as a means of targeting breast tumor cells expressing one or more HER receptors during cancer progression. May be incorporated into the molecule. Further, heregulin molecules are binding determinants for heterodimeric HER receptors containing, for example, a combination of monomers of each of HER-2, -3 or -4. In one embodiment, the monoclonal antibody comprises amino acids 171 to 239 of Heregulin B2 shown in US Pat. No. 5,367,060. As disclosed in US Pat. No. 5,367,060, other portions of Heregulin B2 as well as portions of other Heregulin molecules may be used.
[157] In addition, the antibody or antigen-binding portion thereof of the present invention can be used for the treatment of tumors of the central nervous system. Nestin proteins expressed during fetal development in normal mammals are expressed in tumors of the central nervous system, including most forms of brain cancer (McKay, D. Ronald, US Pat. No. 5,338,839, 1994). 16 August). Nestin is also expressed in melanoma of the skin and melanoma metastasized to other organs [V. A. Florenes, R. Holm, O. Myklebost, U. Lendahl, O. Fodstad, Cancer Res. 54: 354-6, 1994], which is difficult to detect and treat. When using the molecules of the present invention in the treatment of brain tumors, the preferred site of delivery is to deliver directly to the central nervous system or directly to the brain via spinal injection or microneedle delivery. For metastatic cancer, the preferred route of delivery is either direct injection into the circulatory system or using the ex vivo blood methods described herein.
[158] Examples of other tumor types that can use the methods of the invention include Wilm's tumor, a pediatric kidney cancer caused by somatic mutations in a single copy of a gene normally found in two intact copies of a patient [AJ Buckler, KM Call, TM Glaser, DA Haber, DE Housman, CY Ito, J. Pelletier, Rose, EA Rose, U.S. Patent No. 5,350,840]. 95% of cases of Wilms' tumor can be surgically cured, and the binder is considered suitable as an adjuvant treatment modality for surgical patients. Other examples of known cancer-related proteins in which the compositions and methods of the materials of the present invention are suitable are those associated with gastrointestinal cancer (International Application WO 95/14085, May 26, 1995, R. Fishel et al.). Proteins, proteins characterized by various drug resistance developments during chemotherapy (US Pat. No. 5,198,344 to JM Croop et al.), Rb , ras and c-myc, and the like, and are known to those of ordinary skill in the art. There are a number of oncogenes available for analysis. The compositions of the present invention are suitable, for example, for the inhibition of secreted enzymes, such as matrix metalloproteinases, which are believed to enhance tumor metastasis. [Liotta, LA, et al., (1991), Cell, 64: 327-336. In embodiments of such enzyme inhibition, binding agents bearing binding determinants for matrix metalloproteinases and other binding determinants for FcαR facilitate the inhibition and removal of activity in situ of these enzymes. something to do. When used in combination with standard surgical methods and chemotherapy, it is expected that cancer recurrence will be reduced and long-term survival will increase.
[159] Effector cells can target autologous disease by targeting lymphocytes producing auto-antibodies, as well as tumor cells, or treat allergies by targeting lymphocytes producing IgE. Autoimmune disorders that can be treated with the binder of the present invention include diabetes mellitus, arthritis (rheumatic arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, etc.), multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis ( Dry cornea conjunctivitis, alopecia areata, allergic reactions caused by arthropod bites, Crohn's disease, aphthous ulcers, iris infections, conjunctivitis, including atopic dermatitis and eczema dermatitis), psoriasis, Sjoegren's Syndrome and Sjogren's syndrome , Keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, skin lupus erythematosus, scleroderma, vaginitis, proctitis, drug rash, leprosy reverse reaction, leprosy nodule erythema, autoimmune uveitis, allergic encephalomyelitis, acute necrotic bleeding brain disorder Bilateral progressive sensorineural hearing loss, aplastic anemia, pure red blood cell emptying , Idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic hepatitis, Stevens-Johnson syndrome, idiopathic sprue, squamous gland, Crohn's disease, Graves' eye disease, sarcoidosis, primary biliary cirrhosis, posteroencephalitis and interstitial pulmonary fibrosis have. Thus, autoimmune diseases can be treated, for example, by administering to a subject suffering from an autoimmune disease an antibody having one or more antigen binding regions specific for human CD89 and an antigen binding region specific for epitopes on autoimmune cells. In one embodiment, the target epitope is an epitope on self-antibody. Thus, resting B lymphocytes with self-antibodies on their surface are targeted and destroyed by multispecific antibodies linking B lymphocytes and effector cells expressing CD89, thereby inducing effector cell function leading to lysis of B lymphocytes. Can be.
[160] Similarly, antibodies having one or more binding specificities for CD89 and one or more binding specificities for epitopes on lymphocytes producing IgE can be used for the treatment of allergy. In addition, the antibody used to treat allergy in a subject may be a binder having one or more antigen binding regions specific for epitopes on the IgE antibody. Thus, such binding agents can lyse target cells by linking effector cells expressing CD89 and target cells that are coated with IgE, such as basophils and mast cells. Since such treatment can prevent the antigen from binding to the IgE molecule, these cells can also prevent the secretion of allergic mediators such as histamine. In addition, the antibody may bind to soluble IgE, thereby preventing IgE from binding to mast cells and basophils.
[161] The target may also be a microorganism (bacteria or virus) or soluble antigen (eg rheumatoid like factor or other autoantibodies and toxins). Microorganisms include pathogens such as viruses, bacteria, fungi, protozoa and the like. Microorganisms may also be targeted by targeting cells infected by the microorganism, eg, cells infected with a pathogen.
[162] Thus, the present invention, for example, administers to a subject suffering from an infectious agent an effective amount of a bispecific molecule of the invention having at least one antigen binding region specific for CD89 and at least one antigen binding region specific for epitopes on microorganisms. Thereby providing a method of treating an infection. The term “infectious disease” is meant to encompass disorders caused by one or more bacteria, viruses, fungi and protozoa, and the organisms that cause such diseases are collectively referred to as “pathogens”. In the present invention, examples of pathogens include Mycobacterium tuberculosis , Mycobacterium leprae , Pseudomonas aeruginosa , Shigella dysenteria ), Salmonella typhi , Salmonella paratyphi ( S. paratyphi ), Staphylococcus aureus , Streptococcus hemolyticus , Hemophilus pneumoniae , the Sherry Bahia coli (Escherichia coli) serotype O157, chlamydia (Chlamydia) species, Helicobacter pylori (Helicobacter) species, HIV-1, -2 and -3, HTLV, FELV, HSV-I and -II, hepatitis B virus (Eg HBV major surface antigen), non-A non-B non-B hepatitis C virus, Epstein-Barr virus (EBV glycoprotein), pox virus, rabies virus; Aspergillus species; Ent amoeba Heath Tolly urticae (Entamoeba histolytica), jiahreu Dia (Giardia) species; Newcastle Disease Virus; Toxoplasma Oregon diimide (Toxoplasmagondii); And Candida albicans , and the like, but are not limited thereto. Methods of obtaining in vitro unique epitopes from these organisms through protein screening and peptide analysis are well known to those skilled in the art. Thus, preferred antibodies of the present invention possess one or more antigen binding regions specific for epitopes on any of these microorganisms.
[163] In a preferred embodiment, the antibody has an antigen binding region specific for the envelope glycoprotein of the HIV virus, eg, gp41 of HIV. Antibodies specific for gp120 or CD4 are also within the scope of the present invention. In one embodiment, said antibody is derived from human anti-HIV-1 IgG1 mAb, DZ33.
[164] IgA plays an important role in mucosal defense and CD89 is found on effector cells in mucosal regions such as monocytes and macrophages (see, eg, Shen, L. and Collins, J. (1989) Immunology 68: 491). For example, monocytes and macrophages on mucosal surfaces in the lung have been shown to express CD89 (Shen, L. and Collins, J. (1989) Immunology 68: 491). Accordingly, the present invention provides a method for removing microorganisms or any unwanted cells from mucosal areas. This method is particularly useful in that the mucosal site is often the entry point for invading organisms, and further that the peroxide is a potent microbial agent. For example, oxygen metabolites such as superanions have been shown to have bactericidal and bacteriostatic effects.
[165] The antigen binding region for the target epitope may be a ligand for a receptor, eg, a growth factor or differentiation factor, and may target a binding agent to cells bearing a receptor for these growth factors or differentiation factors. For example, an antibody of the present invention may comprise at least some or modified forms thereof capable of interacting with epidermal growth factor (EGF) or epidermal growth factor receptor (EGF-R). The antibody may also comprise a binding portion of heregulin. In another preferred embodiment of the invention, the ligand is a small peptide such as bombesin, gastrin-releasing peptide (GRP), ritorin, neuromedin B or neuromedin C. Sequences of such peptides can be found, for example, in US Pat. No. 5,217,955 and the like, the contents of which are incorporated herein by reference. The ligand may be a modified form of any of these peptides. The modification may increase binding to the receptor, may reduce binding to the receptor, or may not affect binding to the receptor. In addition, ligand modification may convert agonists into antagonists, which may inhibit rather than stimulate cell proliferation. Ligand modifications can be additions, deletions, substitutions or modifications of one or more amino acids.
[166] Effector cells for targeting may be human leukocytes, preferably macrophages. Other cells include monocytes or other IgA-receptor bearing cells. If necessary, effector cells can be obtained from the host to be treated.
[167] Targeted effector cells, ie effector cells coated with a binder of the present invention, can be administered as a cell suspension in a physiologically acceptable solution. The number of cells administered can be about 10 ⁸ to 10 ⁹ , but will vary depending on the purpose of treatment. In general, this will be an amount sufficient to determine location in the target cell and kill the target cell via antibody dependent-mediated cytolysis (ADCC). The route of administration may also vary. For example, in tumor therapy, targeted effector cells may be administered intravenously or directly at the site of the tumor, depending on the location of the tumor, for example in the case of ovarian carcinoma, directly into the abdominal cavity.
[168] Bispecific and multispecific molecules of the invention may further comprise a third binding specificity. In one embodiment, the third binding specificity is a molecule that increases the immune response to the target cell by binding to an anti-enhancing factor (EF) moiety, for example a surface protein involved in cytotoxic activity. An “anti-enhancing factor moiety” can be an antibody, functional antibody fragment or ligand that enhances the effect of a binding determinant on an Fc receptor or target cell antigen by binding to a given molecule, eg an antigen or a receptor. An "anti-enhancing factor moiety" can bind an Fc receptor or target cell antigen. Alternatively, the anti-enhancing factor moiety may bind to an entity different from the entity to which the first and second binding specificities bind. For example, the anti-enhancing factor moiety binds to cytotoxic T-cells (e.g., CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cells causing an increased immune response to target cells). Can be combined).
[169] In one embodiment, the bispecific molecules and multispecific molecules of the invention are one or more antibodies or combinations thereof as binding specificities, such as, for example, Fab, Fab ', F (ab') ₂ , Fv or single chain Fv, and the like. Antibody fragments. As described in US Pat. No. 4,946,778, issued to Ladner et al. On August 7, 1990, the antibody may be any minimum fragment thereof, such as a light or heavy chain dimer or an Fv or a single chain construct, or the like. , Which is hereby incorporated by reference in its entirety.
[170] While human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.
[171] Chimeric mouse-human monoclonal antibodies (ie, chimeric antibodies) can be prepared by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is cleaved with a restriction enzyme to remove the region encoding the murine Fc, and an equivalent portion of the gene encoding the human Fc constant region (European Patent Application No. 171,496 to International Patent Publication No. PCT / US86 / 02269 of Robinson et al., European Patent Application No. 184,187 to Akira et al., Taniguchi, M.). , European Patent Application No. 173,494 to Morrison et al., International Application WO 86/01533 to Neuberger et al., US Patent No. 4,816,567 to Cabilly et al., European Patent Application No. 125,023 to Cavili et al. Better et al. (1988) Science 240: 1041-1043, Liu et al. (1987) PNAS 84: 3439-3443, Liu et al., 1987, J. Immunol. 139: 3521 -3526, Sun et al. (1987) PNAS 84: 214-218, Nishimura et al., 1987, Canc. Res. 47: 999-1005, Wood et al. (1985) Nature 314: 446-449 and Shaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559).
[172] Chimeric antibodies can be further humanized by replacing sequences of Fv variable regions that are not directly involved in antigen binding with equivalent sequences of human Fv variable regions. General considerations on humanized chimeric antibodies are given in Morrison, SL, 1985, Science 229: 1202-1207 and Oi et al., 1986, BioTechniques 4: 214. These methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of an immunoglobulin Fv variable region from one or more of the heavy or light chains. Sources of such nucleic acids are known to those skilled in the art and can be obtained, for example, from hybridoma 7E3 producing anti-GPII _b III _a antibodies. Thereafter, the recombinant DNA encoding the chimeric antibody or fragment thereof can be cloned into an appropriate expression vector. Alternatively, suitable humanized antibodies can be produced by CDR substitutions (US Pat. No. 5,225,539, Jones et al. 1986 Nature 321: 552-525, Verhoeyan et al. 1988 Science 239: 1534) and [ Beidler et al. 1988 J. Immunol. 141: 4053-4060.
[173] All CDRs of a particular human antibody may be replaced with at least a portion of a CDR that is not a human CDR, or only a portion of the CDRs may be replaced with a CDR that is not a human CDR. Only humanized antibodies need to replace as many CDRs as required to bind the Fc receptor.
[174] An antibody can be humanized via any method that can replace at least a portion of a human antibody CDR with a CDR derived from an antibody that is not a human antibody. Winter describes a method that can be used to prepare humanized antibodies of the invention in British Patent Application GB 2188638A, filed March 26, 1987, which is incorporated by reference in its entirety herein. do. Oligonucleotide position-directed mutagenesis described in international application WO 94/10332 (named Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes) can be used to replace human CDRs with non-human CDRs. .
[175] In addition, chimeric and humanized antibodies in which certain amino acids are substituted, deleted or added are also within the scope of the present invention. In particular, preferred humanized antibodies have amino acid substitutions within the framework regions that, for example, improve binding to the antigen. For example, in humanized antibodies with mouse CDRs, amino acids located within the human framework region may be replaced with amino acids located at the corresponding sites of the mouse antibody. Such substitutions are known to improve the binding of humanized antibodies to antigens in some cases. An antibody wherein an amino acid has been added, deleted or substituted is referred to as a modified antibody or an altered antibody.
[176] In addition, the term modified antibody is meant to include antibodies such as monoclonal antibodies, chimeric antibodies and humanized antibodies, for example, which have been modified by deletion, addition or substitution of a portion of the antibody. For example, an antibody can be modified by deleting the constant region of the antibody and replacing it with a constant region that increases the half-life of the antibody, such as serum half-life, stability or affinity. If through any modification the bispecific and multispecific molecules retain one or more antigen binding regions specific for CD89 and trigger one or more effector functions, such any modification is within the scope of the present invention.
[177] Bispecific and multispecific molecules of the invention are described in chemical techniques (see, eg, DM Kranz et al. (1981) Proc. Natl. Acad Sci. USA 78: 5807), “polydoma”. "Technology (see US Patent No. 4,474,893 to Reading) or recombinant DNA technology.
[178] In particular, bispecific molecules and multispecific molecules of the present invention are known in the art and employ the methods described in the Examples section provided herein using component binding specificities such as anti-target cell binding specificity and anti-CD89. It can be prepared by conjugation of binding specificity. For example, each binding specificity of bispecific and multispecific molecules can be generated separately and then conjugated to each other. If the binding specificity is a protein or peptide, various coupling or crosslinking agents can be used to covalently conjugate. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimalee Mead (oPDM), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate ( Sulfo-SMCC) and the like (Karpovsky et al. (1984) J. Exp. Med. 160: 1686), Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132), Brennan et al. (Science (1985) 229: 81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375) Preferred binders are SATA and sulfo-SMCC, all of which are Pierce Chemical Co. (Lock, Illinois, USA). Ford).
[179] If the binding specificity is an antibody (eg two humanized antibodies), they can be conjugated via sulfhydryl binding of the two heavy chain C-terminal hinge regions. In a particularly preferred embodiment, the hinge region is conjugated after modifying it to contain an odd number, preferably one, sulfhydryl residue.
[180] Alternatively, both of these binding specificities can be encoded in the same vector to be expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are mAb x mAb, mAb x Fab, Fab x F (ab ') ₂ or Ligand x Fab fusion proteins. Bispecific and multispecific molecules of the invention, for example bispecific molecules, are single-chain bispecific, including single-chain molecules such as single-chain bispecific antibodies, one single-chain antibody and binding determinants. Red molecule, or a single chain bispecific molecule comprising two binding determinants. In addition, bispecific molecules and multispecific molecules may be single-chain molecules or may comprise two or more single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in US Pat. Nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653 5,258,498 and 5,482,858 and the like.
[181] The binding of bispecific and multispecific molecules to their specific targets can be determined by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg growth inhibition) or Western This can be confirmed by Western Blot analysis. Typically, each of these assays detects the presence of the protein-antibody complex of interest by using labeled reagents (eg, antibodies) specific for the particular complex of interest. For example, FcR-antibody complexes can be detected using, for example, enzyme-linked antibodies or antibody fragments that recognize and specifically bind antibody-FcR complexes. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in radioimmunoassay (RIA) (e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on, herein incorporated by reference). Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioisotope can be detected by the γ counting method, the scintillation counting method, or by the self-radiating method.
[182] IV. Binding analysis
[183] Various assays can be performed to reveal that the antibody or antigen-binding portion thereof of the invention specifically binds to CD89, such as human CD89. For example, binding assays can be performed that compare the binding of the antibody to the binding of IgA to various cells, which bind to some cells (eg monocytes and macrophages), and some cells (eg For example, K-562 cells). Antibodies of the invention must bind substantially the same set of cells as IgA. Competitive binding experiments with the antibody and IgA will indicate whether both agents recognize the same epitope. Binding experiments can be flow cytometry experiments, indirect immunofluorescence assays or ELISAs, among others. In addition, IgA and the antibody must immunoprecipitate proteins of similar molecular weight from the same cell. In addition, preliminary removal of cell lysates containing CD89 with IgA should result in less protein immunoprecipitation using antibodies than in the absence of such preliminary clearance. Similarly, preliminary removal of cell lysates with the antibody should result in less protein immunoprecipitation using IgA than in the absence of such preliminary clearance. Other tests to prove that the antibody or antigen binding portion thereof of the invention and IgA recognize the same antigen are known in the art. In addition, since CD89, such as human CD89, has been cloned, the binding of antibody to CD89 can be demonstrated using recombinantly produced CD89 or a portion thereof or using cells transfected to express CD89.
[184] In one embodiment, when an antibody of the invention connects a target cell to an effector cell, the antibody stimulates phagocytosis of the target cell by the effector cell. For example, preferred bispecific antibodies of the present invention having one or more antigen binding regions specific for CD89 and one antigen binding region for epitopes on target cells can induce phagocytosis of target cells. Indeed, heterologous antibodies of My43 (mouse anti-CD89 antibody) linked to anti-erythrocyte F (ab) ' ₂ have been shown to mediate erythroid phagocytosis by monocytes.
[185] Phagocytosis assays can be performed as follows: 20 μl of F (ab ′) ₂ -Ig conjugate at a pre-measured concentration of packed target cells, eg, ox erythrocytes (OE) (10 μl). And mix at 10 ° C. for 16 hours to ensure maximum rosette formation. The OE coated with the heterologous antibody is washed to adjust to 4 x 10 ⁷ cells / ml and mixed with equal volume of 2 x 10 ⁶ cells / ml of bone marrow cells. The mixture is incubated at 37 ° C. for 10 minutes, the cells are pelleted and further incubated for 20 minutes before the unremoved OE is dissolved at 4 ° C. with ammonium chloride buffer. Phagocytosis can be analyzed by microscopic examination of cytospin preparations stained with Wright's Giemsa (Sigma). More than 200 cells are counted in two slides. Phagocytosis can be quantified as percentage of cells containing one or more removed red blood cell (s).
[186] Preferred antibodies trigger oxidative rupture, ie the production of peroxide anions in effector cells after CD89 binds to effector cells. For example, My43 (mouse anti-CD89 antibody) has been shown to trigger the production of peroxide anions by U937 cells treated with interferon-γ.
[187] Peroxide analysis can be performed in the solid phase or in solution. Solid phase peroxide analysis can be performed as follows: 100 μg / ml in PBS per well in a flat bottom 6-well PVC tissue culture plate (Falcon Plastics Baxter Corporation, Bedford, Mass.). 1 ml of poly-L-lysine (Sigma, St. Louis, MO) can be treated for 30 minutes at room temperature. Aspirated to dryness, glutaraldehyde (2%, Sigma) can be added and incubated for 15 minutes at room temperature. After washing four times with PBS, 1 ml of PBS containing the desired amount of Ig can be added to incubate for 2 hours at room temperature. The wells can then be washed with PBS, added 100 mM glycine / 0.1% BSA in PBS, and then incubated at 4 ° C. for 18 hours. These were then washed twice with PBS and once with Krebs Ringer Hepes buffered salt solution (KRH) to wash 3 x 10 ⁶ cells / well, KCN 1 mM and Equine Cardiac Peryrictochrome c (Sigma). Product) may be added in KRH containing 1 mg / ml. The cells are spun at 100 xg for 5 minutes to collect on plates and incubated at 37 ° C. PMA (Sigma Product) is added to the positive control at the indicated final concentration. After 30 minutes, the contents are removed, rotated, and the supernatant can be read at 550 nm on a Dynatech spectrophotometer (Dynatech Labs, Chantilly, VA).
[188] Suspension peroxide assays can be performed as follows: Cells are incubated with mAbs for 45 minutes at 4 ° C., then washed in KRH, containing 1 mg / ml KCN and 1 mg / ml of horse heart ferricitochrome c (Sigma). Incubation at 37 ° C. can be initiated by resuspension in the prepared KRH. PMA (Sigma Product) can be added to the positive control at the indicated concentrations. After 30 minutes, samples are removed, spun up and the supernatant can be read at 550 nm on a Dynatech spectrophotometer (Dynatech Labs).
[189] V. Antibody Conjugates / Immunotoxins
[190] In another aspect, the invention features a human anti-CD89 monoclonal antibody or fragment thereof conjugated to a therapeutic component, such as a cytotoxin, drug or radioisotope. When conjugated to cytotoxins, these antibody conjugates are referred to as “immunotoxins”. Cytotoxins or cytotoxic agents include any agent that is harmful to the cell (eg, kills the cell) and the like. Examples include Taxol, Cytokalacin B, Gramicidine D, Ethidium Bromide, Emethine, Mitomycin, Etoposide, Tenofoside, Vincristine, Vinblastine, Colchicine, Doxorubicin, Daunorubicin, Dihydroxy Anthracene dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and furomycin and their analogs or analogs. Therapeutic agents include metabolic inhibitors (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechloretamine, thioepa) Chlorambucil, Melphalan, Carmustine (BSNU) and Romustine (CCNU), Cyclotosamide, Busulfan, Dibromomannitol, Streptozotocin, Mitomycin C and Cis-Dichlorodiamine Platinum (II) ( DDP) cisplatin), anthracycline (e.g. daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin , Mitramycin and anthracycin (AMC) and mitosis inhibitors (eg, vincristine and vinblastine), and the like. Antibodies of the invention can be conjugated to radioisotopes, such as radioactive iodine, to produce cytotoxic radiopharmaceuticals for the treatment of CD89-related disorders, such as cancer.
[191] The antibody conjugates of the present invention can be used to modify a given biological response, and drug components should not be considered limited to typical chemotherapeutic agents. For example, the drug component may be a protein or polypeptide possessing the desired biological activity. Examples of such proteins include enzymatically active toxins or active fragments thereof such as abrin, lysine A, Pseudomonas exotoxin or diphtheria toxin; Proteins such as tumor necrosis factor or interferon-γ; Or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage populations Stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
[192] Techniques for conjugating such therapeutic ingredients to antibodies are known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds .), pp. 243-56 (Alan R. Liss, Inc. 1985), Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds. ), pp. 623-53 (Marcel Dekker, Inc. 1987)], Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. ( eds.), pp. 475-506 (1985)], "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds .), pp. 303-16 (Academic Press 1985) and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982 )]).
[193] VI. Pharmaceutical composition
[194] In another aspect, the present invention provides a composition, eg, a pharmaceutical composition, containing one or a combination of human monoclonal antibodies or antigen-binding portion (s) thereof of the present invention and formulated with a pharmaceutically acceptable carrier. do. In a preferred embodiment, the composition comprises a combination of several (eg two or more) isolated human antibodies or antigen binding portions thereof of the invention. Preferably, each antibody or antigen binding portion thereof in the composition binds to another preselected epitope of CD89.
[195] In one embodiment, the composition is one or a combination of bispecific molecules or multispecific molecules of the invention (eg, retain one or more binding specificities for target cells and one or more binding specificities for CD89) It includes.
[196] In addition, the pharmaceutical compositions of the present invention may be administered in combination therapy, ie in combination with other agents. For example, the combination therapy may comprise a composition of the present invention in combination with one or more anti-tumor agents or other conventional therapies.
[197] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). Depending on the route of administration, the active compounds, ie antibodies, bispecific molecules and multispecific molecules, may be coated with substances that protect the compound from the action of acids and other natural conditions that may inactivate the compound.
[198] “Pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the parent compound and do not confer any unnecessary toxicological effects (see, eg, Berge, SM, et al. (1977). J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, as well as salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphorous acid. Salts derived from acids, phenyl substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic sulfonic acids, aromatic sulfonic acids and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium and calcium, as well as nontoxic organic amines such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloro Salts derived from procaine, choline, diethanolamine, ethylenediamine, procaine and the like.
[199] The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode of administration will vary depending upon the desired result. Active compounds can be prepared with controlled release formulations, including carriers that protect the compound against rapid release, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or commonly known to those skilled in the art. See, eg, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[200] In order to administer a compound of the present invention via a particular route of administration, it may be necessary to coat the compound with a substance that prevents the inactivation of the compound, or to co-administer the compound with the substance. For example, the compound may be administered to a subject in a suitable carrier, such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include conventional liposomes as well as water-in-oil CGF emulsions [Strejan et al. (1984) J. Neuroimmunol. 7:27].
[201] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of injectable sterile solutions or dispersions. The use of such media and agents in pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[202] Typically, the therapeutic composition should be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome or other regular form of construct suitable for high drug concentration. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, it is possible to maintain proper fluidity by using a surfactant. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Agents that delay absorption, such as monostearate salts, gelatin, and the like, can be included in the injectable compositions to prolong its absorption time.
[203] Injectable sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Typically, 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. In the case of sterile powders for the preparation of injectable sterile solutions, preferred methods of preparation are vacuum drying and freeze-drying (freezing) to produce powders of the components from the powder of the active ingredient and any desired further component solution that has been sterile filtered in advance. Dry).
[204] The mode of administration is adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, and the dose may be divided over time into several doses, or the dose may be proportionally reduced or increased as indicated in an emergency treatment situation. It may be. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and for administration of uniform dosages. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit having a predetermined amount of active compound and the required pharmaceutical calculated to produce the desired therapeutic effect. It contains a carrier. The unit dosage form of the invention depends on the inherent properties of the active compound and the specific therapeutic effects to be achieved and (b) the limitations inherent in the art of combining such active compounds for the treatment of sensitivity in the subject, It depends directly on this.
[205] Examples of pharmaceutically acceptable antioxidants include (1) water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite and sodium sulfite, and (2) fat soluble antioxidants such as ascorbyl palmi Tate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate and alpha-tocopherol and the like and (3) metal chelating agents such as citric acid, ethylenediamine tetraacetic acid ( EDTA), sorbitol, tartaric acid and phosphoric acid.
[206] For therapeutic compositions, the formulations of the present invention include formulations suitable for oral, nasal, topical (such as buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject to be treated and the particular mode of administration. Typically, the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will be that amount of the composition which produces a therapeutic effect. Typically, based on 100%, the active ingredient in this amount will range from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%. .
[207] In addition, preparations of the present invention suitable for intravaginal administration include vaginal preparations, tampons, creams, gels, pastes, foams, sprays, and the like, which contain a carrier or the like which is known to be suitable in the art. Dosage forms for topical or transdermal administration of the compositions of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.
[208] As used herein, the phrases “parenteral administration” and “parenteral administration” refer to a mode of administration, usually by injection, that is not enteral and topical administration, including intravenous, intramuscular, Intraarterial, intradural, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transcheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, dural External and intrasternal injections and injections, and the like, but is not limited thereto.
[209] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (eg glycerol, propylene glycol and polyethylene glycol, etc.) and their suitable mixtures, vegetable oils such as olive oil and injections. Possible organic esters such as ethyl oleate and the like. For example, by using a coating material such as lecithin, by maintaining the required particle size in the case of dispersion, it is possible to maintain proper fluidity by using a surfactant.
[210] These compositions may include auxiliaries such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be achieved by using sterilization methods (supra) and including various antibacterial and antifungal agents such as parabens, chlorobutanol and phenol sorbic acid and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like into the compositions. Injectable formulations may also include agents that delay absorption, such as aluminum monostearate, gelatin, and the like, to prolong its absorption time.
[211] When a compound of the present invention is administered to humans and animals as a medicament, it is administered alone or contains, for example, 0.01 to 99.5% (more preferably 0.1 to 90%) of the active ingredient together with a pharmaceutically acceptable carrier. It can be administered as a pharmaceutical composition.
[212] Regardless of the route of administration chosen, the compounds of the invention and / or the pharmaceutical compositions of the invention, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms via conventional methods known to those skilled in the art.
[213] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to achieve the amount, composition, and mode of administration of the active ingredient that are effective to achieve the desired therapeutic response for a particular patient and are not toxic to the patient. . The dosage level chosen is the activity of the specific composition of the invention or its ester, salt or amide used, route of administration, time of administration, rate of release of the specific compound used, duration of treatment, other drugs used with the particular composition used, It will depend on various pharmacodynamic factors, including the compound and / or substance, age, sex, weight, condition, general health and similar medical history and similar factors known in the medical field of the patient to be treated.
[214] Any physician or veterinarian of ordinary skill in the art will readily be able to determine and prescribe the effective amount of the pharmaceutical composition required. For example, a doctor or veterinarian can start a dosage of a compound of the invention used in a pharmaceutical composition at a lower level than necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. . In general, a suitable daily dosage of a composition of the invention will be that amount of the compound which is the lowest dosage effective to produce a therapeutic effect. Such effective dosage will generally depend on the factors mentioned above. Administration is preferably intravenous, intramuscular, intraperitoneal or subcutaneous, preferably near the target site. If desired, the effective daily dosage of the therapeutic composition may be divided into two, three, four, five or six or more divided doses administered individually at appropriate time intervals throughout the day, in some cases as unit dosage forms. May be administered. The compounds of the present invention may be administered alone, but are preferably administered as pharmaceutical formulations (compositions).
[215] Therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention is a needleless hypodermic syringe, for example US Pat. No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, Copper It may be administered using the devices disclosed in 4,941,880, 4,790,824 or 4,596,556. Examples of known implants and modules useful in the present invention include US Pat. No. 4,487,603, which discloses an implantable microinjection pump for administering a medicament at a controlled rate, 4,486,194, which discloses a therapeutic device for administering a medicament through the skin. No. 4,447,233, which discloses a medicament infusion pump for delivering a drug at an accurate infusion rate, and 4,447,224, osmotic drug having a multi-chamber compartment, which discloses an infusion device for implantation of a variable flow for continuous drug delivery. 4,439,196, which discloses a delivery system, and 4,475,196, which discloses an osmotic drug delivery system. These patent documents are incorporated herein by reference through this name. Many other such implants, delivery systems, and modules are known to those skilled in the art.
[216] In certain embodiments, human monoclonal antibodies of the invention can be formulated to be appropriately distributed in vivo. For example, the blood brain barrier (BBB) excludes many highly hydrophilic compounds. In order for the therapeutic compounds of the invention to pass through the BBB (if necessary), they can be formulated, for example, in liposomes. See, for example, US Pat. No. 4,522,811, US Pat. No. 5,374,548, US Pat. No. 5,399,331 and the like for the preparation method of liposomes. Since liposomes may optionally include one or more components that are transported to specific cells or organs, they enhance targeted drug delivery (eg, VV Ranade (1989) J. Clin. Pharmacol. 29: 685). Reference). Examples of targeting components include folate or biotin (eg, US Pat. No. 5,416,016 to Low et al., Mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); Antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140, M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptors [Briscoe et al. (1995) Am. J. Physiol. 1233: 134 (other species thereof may include components of the invention as well as formulations of the invention); p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090, K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123, JJ Killion; IJ Fidler (1994) Immunomethods 4: 273). In embodiments, the therapeutic compounds of the invention are formulated in liposomes, and in a more preferred embodiment, the liposomes comprise targeting components In a most preferred embodiment, liposomes are injected by bolus injection The therapeutic compound is delivered near the tumor or site of infection The composition must be fluid enough to be easily injected The composition must be stable under the conditions of manufacture and storage and must be preserved against microbial contaminants such as bacteria and fungi do.
[217] A “therapeutically effective dose” is one that inhibits tumor growth at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, even more preferably at least about 80% relative to an untreated subject. It is preferable. Cancer inhibition by compounds can be assessed in animal model systems in which efficacy in human tumors is predicted. Alternatively, this property of the composition can be assessed by investigating the in vitro inhibition by the compound through assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or alleviate symptoms in a subject. Those skilled in the art will be able to determine the dosage based on factors such as the subject's height, the severity of the subject's symptoms and the particular composition or route of administration chosen.
[218] The composition must be sterile and must be fluid enough to be delivered through a syringe. Carriers may be water as well as isotonic buffered saline solutions, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining a required particle size in the case of dispersion, it is possible to maintain proper fluidity by using a surfactant. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol and sodium chloride in the composition. Agents that delay absorption, such as aluminum monostearate or gelatin, may be included in the injectable composition for long term absorption.
[219] If the active compound is properly protected as described above, the compound can be administered orally, for example with an inert diluent or an absorbable edible carrier.
[220] VII. Uses and Methods of the Invention
[221] Compositions of the invention (eg, human monoclonal antibodies to CD89 and derivatives / conjugates thereof) are useful for in vitro and in vivo diagnostics and treatment. For example, these molecules can be administered to, for example, in vitro or ex vivo culture cells or, for example, cells in vivo in a subject, to treat, prevent or diagnose various disorders. As used herein, the term "subject" is meant to include humans and non-human animals. Preferred human animals include human patients suffering from disorders characterized by the expression of CD89, typically aberrant expression (eg, overexpression) of CD89.
[222] For example, the composition can be used in vitro or in vivo to diagnose a disease mediated by CD89. After detecting the level of CD89 or the level of cells containing CD89 on the membrane surface using antibodies of the invention, the levels can be associated with the symptoms of a particular disease. Alternatively, the antibody can be used to inhibit or block CD89 function, thereby preventing or alleviating the symptoms of a particular disease, thereby implicating CD89 as a mediator of the disease. This can be accomplished by contacting the sample and the control sample with the antibody under conditions that allow complex formation between the anti-CD89 antibody and CD89. Any complexes formed between anti-CD89 antibodies and CD89 in the samples and controls are detected and compared.
[223] In other embodiments, human antibodies of the invention or binding portions thereof can be used to modulate CD89 levels in effector cells, for example, by capturing and eliminating receptors on the cell surface. For this purpose, mixtures of anti-Fc receptor antibodies can also be used.
[224] In addition, methods of treating disorders such as autoimmune disorders, cancer or pathogenic infections, etc. using the bispecific human antibodies and multispecific human antibodies described above are also within the scope of the present invention. Such antibodies include one or more binding specificities for CD89 and one or more binding specificities for an antigen binding region to a target antigen. In other embodiments, the antibody comprises a third binding specificity for the binding specificity of the antigen binding region to various epitopes of the same target antigen and / or receptor. Methods for removing unwanted cells, ie target cells or antigens, from a subject include treating the subject with a bispecific antibody or multispecific antibody of the invention. In one embodiment, the method comprises obtaining an aliquot of a blood or blood cell sample in a subject, wherein said blood or blood cell is in a pharmaceutically acceptable carrier at a therapeutically effective dosage of a bispecific antibody or multispecific of the invention. In vitro treatment with the enemy antibody and returning the treated blood or blood cells back to the subject. It is desirable to isolate and culture the cells of the blood sample and multiply, and more preferably treat the cells of the blood sample with an agent that enhances the number of CD89 or the activity of CD89. Such agents include cytokines, lymphokines or growth factors such as G-CSF, GM-CSF, IFN-γ, TNF and interleukins such as IL-2, IL-10 and IL-12 and the like. .
[225] In another embodiment, the present invention provides a method of immunizing a subject against an antigen found in cancer antigens, pathogens, or cells infected with a pathogen. This method comprises a composition comprising a bispecific or multispecific antibody in a pharmaceutically acceptable carrier, the composition comprising a binding specificity for CD89 and binding specificity for an infectious pathogen or an antigen of an infected cell or an epitope of a cancer cell. Administering. Alternatively, the composition may comprise an anti-CD89 antibody linked to one or more antigens of an infectious pathogen or infected cell or cancer cell.
[226] In other embodiments, a human antibody of the invention that blocks or inhibits IgA binding to CD89 is characterized by a disease characterized by abnormal endogenous IgA, eg, precipitation of circulating IgA-containing complexes and / or IgA-immune complexes. It is used for the treatment of diseases, for example, chronic hepatitis, Henoch-Scholine purpura (HSP), IgA nephropathy (Berber's disease) or IgA-glomerulonephritis. Human anti-CD89 antibodies of the invention can be administered to patients suffering from such disorders to inhibit or downregulate endogenous IgA binding to CD89. For example, although not intended to be limited to mechanisms, CD89 is blocked by the administration of the anti-CD89 antibody of the present invention, resulting in abnormal endogenous IgA being unable to bind to the receptor, thus triggering undesirable effector functions. .
[227] In other embodiments, since the human monoclonal antibodies of the invention have the advantage of not inducing or only inducing minimal complement-mediated cell lysis, the side effects of complement-activated symptoms, such as acne triggers, Even less.
[228] Suitable methods of administration of the compositions of the invention (eg human antibodies, multispecific molecules and bispecific molecules) are known in the art of antibody-based therapy. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. Goldenberg, D. M. et al. (1981) Cancer Res. 41: 4354-4360 and European Patent No. 0265 997, the molecules may be coupled to radionuclides such as 131I, 90Y and 105Rh. It is also possible to couple compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) to infection inhibitors.
[229] The human anti-CD89 antibody or antigen-binding fragment thereof may be co-administered with other therapeutic agents, such as cytokines or chemotherapeutic agents, or co-administered with other known therapies, such as radiation therapy. Such therapeutic agents include, among others, cytokines such as G-CSF, GM-CSF, IL-2, IFN-γ and IFN-α, anti-neoplastic agents such as doxorubicin (Adriamycin), cisplatin bleomycin Sulfates, carmustine, chlorambucil and cyclophosphamide hydroxyurea, etc., which are themselves effective only at levels that are toxic or nearly toxic to patients. Cisplatin is administered intravenously once every four weeks at a dose of 100 mg / m ² , and adriamycin is administered intravenously once every 21 days at a dosage of 60 to 75 mg / m ² . Co-administration of a human anti-CD89 antibody or antigen-binding fragment thereof of the present invention and a chemotherapeutic agent acting through a different mechanism is to provide two anticancer agents that cause a cytotoxic effect on human tumor cells. Such co-administration may solve the problem that the tumor cells do not respond to the antibody by generating drug resistance in the tumor cells or by changing the antigenicity of the tumor cells.
[230] In addition, complement compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) that retain complement binding sites, such as IgG1, -2 or -3 or the portion that binds to complement at IgM. Can also be used in the presence of In one embodiment, ex vivo treatment of a cell population comprising target cells with the binder of the present invention and appropriate effector cells can be supplemented by adding complement or serum containing the complement. By binding the complement protein, phagocytosis of target cells coated with the binding agent of the present invention can be improved. In other embodiments, target cells coated with the compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) may be lysed by complement. In other embodiments, the compositions of the present invention do not activate complement.
[231] In addition, compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) may also be administered in conjunction with complement. Thus, compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement are within the scope of the present invention. Such compositions are advantageous in that the complement is located proximate to the human antibody, multispecific molecule or bispecific molecule. Alternatively, human antibodies, multispecific or bispecific molecules of the invention and complement or serum may be administered separately.
[232] Also included in the scope of the invention are kits comprising compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) and instructions for use. The kit may comprise one or more additional reagents such as complement or the like or one or more additional human antibodies of the invention (e.g., a human antibody that differs from the first human antibody and has complement activity that binds to an epitope of the CD89 antigen). It may further contain.
[233] In addition, the compositions of the invention (eg, human antibodies, multispecific molecules and bispecific molecules) can be used to target cells expressing CD89, for example to label such cells. When so used, the binder can be linked to a molecule that can be detected. Thus, the present invention provides a method for locating cells expressing CD89 ex vivo or in vitro. Detectable labels can be, for example, radioisotopes, fluorescent compounds, enzymes or enzyme cofactors.
[234] In one embodiment, the present invention relates to a method for treating a sample and a control sample with said antibody or antigen-binding portion thereof under conditions that permit the formation of a complex between a human monoclonal antibody or antigen-binding portion thereof that specifically binds to CD89 and CD89. Provided are methods of detecting the presence of a CD89 antigen or measuring the amount of a CD89 antigen in a sample, comprising contacting. The formation of the complex is then detected, where the difference in complex formation between the sample and the control sample is indicative of the presence of the CD89 antigen in the sample.
[235] In another embodiment, the invention provides a method of detecting the presence or quantification of an amount of Fc-expressing cells in vivo or in vitro. The method comprises the steps of (i) administering a composition (eg, multispecific molecule or bispecific molecule) or fragment thereof of the invention conjugated to a detectable marker and (ii) subjecting the subject to the detectable Exposing the marker to a means for detecting the marker to identify the region containing the Fc-expressing cell.
[236] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention thereto. The contents of all figures and references, patent documents, and patent application publications cited in this application specification are hereby incorporated by reference in their entirety.
[237] Example 1: Generation of transgenic (Cmu targeted) mice for preparation of fully human monoclonal antibodies against CD89
[238] Construction of CMD Targeting Vectors
[239] Plasmid pICEmu contains an EcoRI / XhoI fragment of the murine Ig heavy chain locus spanning the mu gene, obtained from the Balb / C genomic lambda phage library [Marcu et al. Cell 22: 187, 1980]. This genomic fragment was subcloned into the XhoI / EcoRI site of plasmid pICEMI9H [Marsh et al; Gene 32, 481-485, 1984]. The heavy chain sequence included in pICEmu extends downstream of the EcoRI site located 3 'adjacent to the mu intron enhancer to the XhoI site located about 1 kb downstream of the last transmembrane exon of the mu gene, but much of the mu switch repeat region. Is this. Deletion by subculture in E. coli .
[240] Targeting vectors were constructed as follows: 1.3 kb HindIII / SmaI fragments were subcloned into pBluescript (Stratagene, La Jolla, Calif.), Cleaved from pICEmu and digested with HindIII / SmaI. This pICEmu fragment extends from the HindIII site located at about 1 kb of Cmu1 5 'to the SmaI site located in Cmu1. The resulting plasmid was digested with SmaI / SpeI and an about 4 kb SmaI / XbaI fragment from pICEmu was inserted, extending from the SmaI site of Cmu1 3 ′ to the XbaI site located downstream of the last Cmu exon. The resulting plasmid pTAR1 was linearized at the SmaI site and a neo expression cassette was inserted. This cassette is under the transcriptional control of the mouse phosphoglycerate kinase (pgk) promoter (XbaI / TaqI fragment, Adra et al. (1987) Gene 60: 65-74), and the pgk polyadenylation site (PvuII / HindIII fragment). , Boer et al. (1990) Biochemical Genetics 28: 299-308). This cassette is obtained from plasmid pKJ1 (described in Tybulewicz et al. (1991) Cell 65: 1153-1163), from which the neo cassette is cleaved as an EcoRI / HindIII fragment and cleaved with EcoRI / HindIII. Subcloning in pGEM-7Zf (+) produces pGEM-7 (KJ1). pGEM-7 (KJ1) was digested with EcoRI / SalI to cleave the neo cassette and blunt ended to subclonal the genome Cmu sequence to the SmaI site of plasmid pTARI. Mansour et al. (1988) Nature 336: 348-352, the resulting plasmids were linearized with NotI and the herpes simplex virus thymidine kinase (tk) cassette was inserted to increase the ES clones carrying homologous recombinants. . See Tybulewicz et al. (1991) Cell 65: 1153-1163, this cassette consists of the coding sequence of the tk gene surrounded by a mouse pgk promoter and a polyadenylation site. The resulting CMD targeting vector contains a homology of about 5.3 kb in total with the heavy chain locus and is designed to produce a mutant mu gene in which a neo expression cassette is inserted at the only SmaI site of the first Cmu exon. The targeting vector was linearized with PvuI cleaving within the plasmid sequence and then electroporated into ES cells.
[241] Generation and Analysis of Targeted ES Cells
[242] In essence, Robertson, EJ (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach (EJ Robertson, ed.) Oxford: IRLPress, p. 71-112, AB-1 ES cells [McMahon, AP and Bradley, A., (1990) Cell 62: 1073-1085] were grown on cell division inactive SNL76 / 7 cell feed layer (same literature). Linearized CMD targeting vectors are described in Hasty et al., Hasty, PR et al. (1991) Nature 350: 243-246, followed by electroporation to AB-1 cells. Electroporated cells were plated in 100 mm dishes at a density of 1 × 10 ⁶ to 2 × 10 ⁶ cells / dish. After 24 hours, G418 (200 μg / active ingredient (mL)) and FIAU (5 × 10 ⁻⁷ M) were added to the medium and drug-resistant clones were allowed to develop over 8-9 days. Clones were picked out and treated with trypsin, split into two parts and further propagated. Then half of the cells derived from each clone were frozen and the other half analyzed for homologous recombination between the vector and the target sequence.
[243] DNA analysis was performed via Southern blot hybridization. Laird et al., Laird, P. W. et al., (1991) Nucleic Acids Res. 19: 4293, DNA was isolated from clones. Isolated genomic DNA was digested with SpeI and detected with probe A (see FIG. 1), a 915 bp SacI fragment, which hybridizes with the sequence between the mu intron enhancer and the mu switch region. Probe A detects a 9.9 kb SpeI fragment from the wild type locus and a diagnostic 7.6 kb band from the mu locus recombined with the CMD targeting vector (neo expression cassette contains the SpeI site). Three of the 1132 G418 and FIAU resistant clones screened through Southern blot analysis showed a 7.6 kb SpeI band indicating homologous recombination at the mu locus. These three clones were further cleaved with the enzymes BglI, BstXI and EcoRI to confirm that the vector was homologously integrated into the mu gene. When hybridized with probe A, wild-type DNA was digested with BglI, BstXI or EcoRI and analyzed by Southern blot to produce fragments of size 15.7 kb, 7.3 kb and 12.5 kb, respectively, but the presence of the targeted mu allele was 7.7 kb, 6.6 kb and 14.3 kb in size fragments. All three positive clones detected by cleavage with SpeI exhibited the expected BglI, BstXI and EcoRI restriction fragments in which the neo cassette was diagnosed as having been inserted into the Cmu1 exon.
[244] Generation of mice carrying mutated mu genes
[245] Bradley et al., Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p. 113-151, three targeted ES clones, designated 264, 272 and 408, were thawed and injected into C57BL / 6J blastocysts. The injected blastocysts were transferred into the female uterus of childbearing status to produce chimeric mice that exhibited a mixture of injected ES cells and cells derived from host blastocysts. The distribution of ES cells in the chimera can be estimated visually by the amount of staining of the agouti envelope derived from the ES cell line on the black C57BL / 6J background. Clones 272 and 408 produced only a low percentage of chimeras (i.e., a low percentage of aguti coloration), while clone 264 produced a high percentage of male chimeras. These chimeras were crossed with C57BL / 6J females to generate Aguti progeny, which directs germline delivery of the ES cell genome. DNA from the tail biopsy was digested with BglI and Southern blot analysis was performed to screen for the targeted mu genes (as described above for analysis of ES cell DNA). About 50% of Aguti progeny showed wild type bands of 15.7 kb as well as hybridized BglI bands of 7.7 kb, demonstrating germline delivery of targeted mu genes.
[246] Analysis of Transgenic Mice for Functional Inactivation of the mu Gene
[247] To determine whether neo cassette insertion into Cmu1 inactivates the Ig heavy chain gene, we cloned the mouse and clone 264 chimeras homozygous for JHD mutations that delete JH gene segments to inactivate heavy chain expression [Chen et al, (1993) Immunol. 5: 647-656. Four Aguti offspring were generated. Serum was collected when these animals were 1 month old and analyzed for the presence of murine IgM by ELISA. Two of the four offspring were completely devoid of IgM (see Table 1). DNA from tail biopsies was subjected to Southern blot analysis by BglI cleavage and hybridization with Probe A (see FIG. 1), and by StuI cleavage and hybridization with 475 bp EcoRI / StuI fragments (same document). By determining the genotype, animals that failed to express serum IgM demonstrated that one allele of the heavy chain locus carries a JHD mutation and the other allele carries a Cmu1 mutation. Mice heterozygous for JHD mutations showed serum Ig at wild-type levels. This data demonstrates that Cmu1 mutations inactivate the expression of the mu gene.
[248] mouseSerum IgM (µg / mL)IgH chain genotype 42<0.002CMD / JHD 43196+ / JHD 44<0.002CMD / JHD 45174+ / JHD 129 x BL6F1153+ / + JHD<0.002JHD / JHD
[249] Table 1 shows mice bearing both CMD and JHD mutations (CMD / JHD), mice heterozygous for JHD mutations, wild-type (129Sv x C57BL / 6J) F1 mice (+ / +) and JHD The levels of serum IgM detected via ELISA for B cell deficient mice (JHD / JHD) homozygous for the mutations are shown.
[250] Example 2: Generation of HCO12 Transgenic Mice for Production of Fully Human Monoclonal Antibodies Against CD89
[251] HC012 human heavy chain transgene
[252] 80 kb insert of pHC2 [Taylor et al., 1994, Int. Immunol., 6: 579-591] and HCO12 transgenes were generated by co-injection of a 25 kb insert of pVx6. Plasmid pVx6 was constructed as described below:
[253] A 8.5 kb HindIII / SalI DNA fragment comprising the human germ line VH1-18 (DP-14) gene with approximately 2.5 kb of 5 'flanking and 5 kb of 3' flanking genomic sequence was identified by plasmid vector pSP72 (Wisconsin, USA). Subcloning in Promega, Madison) produced plasmid p343.7.16. A 7 kb BamHI / HindIII DNA fragment comprising the human germ line VH5-51 (DP-73) gene with about 5 kb of 5 'flanking and 1 kb of 3' flanking genomic sequence was identified by pBR322 plasmid cloning vector pGP1f. Taylor et al. 1992, Nucleic Acids Res. 20: 6287-6295 to generate plasmid p251f. A new cloning vector pGP1k derived from pGP1f was digested with EcoRV / BamHI to comprise a 10 kb containing human germline VH3-23 (DP47) gene with about 4 kb of 5 'flanking and 5 kb of 3' flanking genomic sequence. Ligation to EcoRV / BamHI DNA fragments. The resulting plasmid p112.2RR.7 was digested with BamHI / SalI and ligated with purified 7 kb BamHI / SalI insert of p251f. The resulting plasmid pVx4 was digested with XhoI and ligated with the 8.5 kb XhoI / SalI insert of p343.7.16.
[254] Clones containing the VH1-18 gene in the same direction as the other two V genes were obtained. This clone, named pVx6, was then cleaved with NotI and Hogan et al. As described in Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual, 2 ^nd edition, 1994, Cold Spring Harbor Laboratory Press, Plainview, NY, a purified 26 kb insert was inserted with a purified 80 kb NotI of pHC2. The sieve was co-injected into the pronucleus of a 1/2 day (C57BL / 6J × DBA / 2J) F2 embryo at a molar ratio of 1: 1. Three independent lines of transgenic mice were established that included sequences from both Vx6 and HC2 from mice developing in injected embryos. These strains were named (HC012) 14881, (HC012) 15083 and (HC012) 15087. Each of these three lines were then subjected to the CMD mutation, JKD mutation described in Example 1 [Chen et al. 1993, EMBO J. 12: 811-820 and (KCo5) 9272 transgene [Fishwild et al. 1996, Nature Biotechnology 14: 845-851]. The resulting mice expressed human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of endogenous mouse heavy chain locus and kappa light chain locus.
[255] Example 3: Production of Human Monoclonal and Bispecific Antibodies Against CD89
[256] Human anti-CD89 monoclonal antibodies were generated by immunizing HC012 mice with recombinant soluble CD89 antigen.
[257] In particular, HC012 mice were first immunized with 20 μg of an emulsified antigen in Freund's complete adjuvant, followed by two or more times in Freund's incomplete adjuvant. Mice were immunized via intraperitoneal injection every two weeks.
[258] Ten days after the last immunization, human IgG anti-CD89 titers were determined via ELISA (as described below). Mice that develop sufficient titers (anti-CD89 IgG response) were IV boosted with 23 μg antigen and splenectomy was performed after days 3 and 2. Spleens from the responding mice were harvested and dispersed into single cells.
[259] To generate hybridomas that produce anti-CD89 antibodies, splenocytes of mice bearing plasma containing anti-CD89 antibodies were transferred to P3X63-Ag8.653 cells (ATCC with Accession No. ATCC CRL 1580). Mouse myeloma cells) and PEG. After growing the hybridomas, each well containing the hybridomas was screened for production of human IgG using an anti-human IgG ELISA.
[260] Positive hybridomas were screened based on the following characteristics: (1) production of human IgG1κ antibody, (2) binding to soluble recombinant CD89 and (3) binding to CD89-expressing immune cells. Three positive clones, named 7.4 (7F12), 8.2 (8D2) and 14.1 (14A8), were found to meet this criterion.
[261] Binding assays were based on solid ELISA based assays. Briefly, soluble recombinant CD89 (1 μg / ml) in PBS was coated onto 96-well microtiter plates and incubated overnight at room temperature. The plates were then washed with PBS-0.2% Tween-20 (PBST) and blocked with chicken serum albumin in PBST for 1 hour at room temperature. Supernatants or purified antibodies (in PBS) from hybridomas producing anti-CD89 were added to the wells and incubated for 2 hours at room temperature. Plates were washed with PBST as above and then incubated with 50 μl of HRP-conjugated goat-anti-human for 1 hour at room temperature. After washing, 150 mg of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) in 500 mL of 0.1 M citric acid, pH 4.35) / H ₂ 0 ₂ (30% per 10 mL of ABTS solution) 100 μl of H ₂ 0 ₂ ) chroman / substrate solution was added to each well and samples were read using a microplate reader at a wavelength of 405 nm.
[262] Hybridomas producing human IgG bound to CD89 were further characterized when analyzed by ELISA (human kappa) for isotype (same protocol as described above).
[263] Three cell lines 7.4, 8.2 and 14.1 were selected based on the production of antibodies that bind to soluble CD89 bearing the human IgG1κ isotype. Cellmax bioreactor (Laguna Hills, Calif.), Using HyQ-CCM1 medium containing 1-5% Fetalclone (Hyclone, Logan, Utah) Antibodies were isolated from Cellco and purified by column chromatography. When labeled using fluorescent isothiocyanate (FITC), purified monoclonal antibodies were dialyzed globally against 0.3 M sodium carbonate buffer, pH 9.5. A FITC stock solution was prepared by dissolving 1 mg of solid FITC in 1 ml of DMSO. Mixing was continued with dropwise addition of the FITC stock solution in an amount providing 50 μg FITC per mg of antibody protein. After addition of FITC, the solution was incubated under dark conditions for 1-3 hours at room temperature. FITC labeled antibodies were isolated by gel filtration on Sephadex G-10 columns equilibrated in PBS.
[264] As described in the Examples below, purified antibodies had additional features related to binding specificity and activity.
[265] Example 4: Characterization of Human Monoclonal Antibodies to CD89
[266] I. Combined / Specific Studies
[267] Purified monoclonal antibodies (Mab) (eg 7.4, 8.2 and 14.1) from hybridoma supernatants showing significant binding to CD89 when detected by ELISA were subjected to flow cytometry (FACS assay) as described below. And further tested and confirmed binding to the following CD89 expressing cells: (1) murine 2a1.6 cells transformed to express human CD89 (“2a1.6 / CD89 cells”), (2) human PMNL cells (“HuPMN”) and (3) PMNL cells expressing human CD89, isolated from transgenic mice (“FcαRITg MsPMN”).
[268] In particular, MAb 14.1 has been found to recognize CD89 (FcαRI) in extracellular domain 2 at a position other than the IgA-binding site, which blocks known murine anti-CD89 Mab, eg, FcαRI ligand-binding domains. It is distinguished from My43 (mouse IgM) (Shen, L. et al., 1989 R Immunol. 143: 4117).
[269] PMNL Isolation
[270] Human PMNL was isolated from heparin treated venous blood of healthy volunteers via Ficoll-Histopaque (Sigma) density gradient centrifugation. PMNL purity measured by the cell rotator preparation was greater than 95% and cell viability was greater than 98% (measured by trypan blue exclusion).
[271] Prior to isolating murine PMNL, mice were injected subcutaneously with 15 μg of polyethylene-glycol granulocyte colony-stimulating growth factor (PEG-G CSF, kidney obtained from Dr. J. Anderson, Thousand Oaks Amgen, California, USA). PMNL number was increased. After 3 days, blood was collected from the retro-orbital plexus. After red blood cells were removed via storage hemolysis, the remaining white blood cells were washed three times using RPMI 1640 medium supplemented with 10% FCS (Gibco BRL, Grand Island, NY). . FACS analysis was performed on FACSsan (Becton Dickinson, San Jose, Calif.), With leukocytes containing about 55% PMNL, about 40% lymphocytes, about 3% monocytes, and about 1% It was confirmed that it consists of eosinophils. Cell viability, as determined by trypan blue exclusion, was always greater than 95%.
[272] FACS Analysis 1 (Combination Study)
[273] 50 μl of antibody dilution was added to 50,000 2a1.6 / CD89 cells in 50 μl of Facs buffer and incubated at 4 ° C. for 30 minutes. The cells were then washed three times with 100 μl of Facs buffer and incubated with rabbit (Fab ₂ ) anti-Hu IgG-FITC for 30 min at 4 ° C. and washed three times with 100 μl of Facs buffer. Cells were resuspended in 200 μl of Facs buffer. Fluorescence was measured on Facscalibur (Becton Dickinson).
[274] FACS analysis 2 (specificity study 1)
[275] 25 μl of 1 mg / ml in Facs buffer was added to 50,000 2a1.6 / CD89 cells in 50 μl of Facs buffer and incubated at 4 ° C. for 15 minutes. 25 μl of antibody dilution was added to incubate for 30 minutes at 4 ° C. and washed three times with 100 μl of Facs buffer. Cells were then incubated with rabbit (Fab ₂ ) anti-Hu IgG-FITC for 30 minutes at 4 ° C., washed three times with 100 μl of Facs buffer and resuspended in 200 μl of Facs buffer. Fluorescence was measured on a faxcaliber (Becton Dickinson).
[276] FACS analysis 3 (specificity study 2)
[277] 50 μl of antibody dilution was added to the control cells Jurkat / IIIa, CHO / IIIbNA.2, CHO / IIIbNA.1, 2a1.6 / CD64 or SLC cells as well as 50,000 2a1.6 / CD89 cells in 50 μl of Facs buffer. Incubated at 4 ° C. for 30 min and washed three times with 100 μl of Facs buffer. Cells were then incubated with rabbit (Fab ₂ ) anti-Hu IgG-FITC for 30 minutes at 4 ° C. and washed three times with 100 μl of Facs buffer. Cells were resuspended in 200 μl of Facs buffer. Fluorescence was measured on a faxcaliber (Becton Dickinson).
[278] FACS analysis 4 (whole blood binding study)
[279] 50 μl of antibody dilution was added to 50 μl of human, mouse or CD89 Tg mouse whole blood and incubated at 4 ° C. for 30 minutes. Cells were lysed with Facs lysis buffer and incubated at 4 ° C., washed three times with 100 μl of Facs buffer, incubated with rabbit (Fab ₂ ) anti-Hu IgG-FITC for 30 minutes at 4 ° C. and 100 μl of Facs buffer. Wash three times. Cells were then resuspended in 200 μl of Facs buffer. Fluorescence was measured on a faxcaliber (Becton Dickinson).
[280] II. Competition research
[281] Mab 14.1 and 7.4 were tested for binding inhibition of human IgA and Mab My43 (mouse anti-CD89 antibody) and CD89 via blocking studies and inhibition ELISA as described below.
[282] MY43, like IgA, competes with IgA binding because it binds to CD89 at its natural ligand binding site. In addition, Mab 14.1 and 7.4 were also tested for binding inhibition of Mab A77 and A59 (they bind at positions other than the binding site of the natural ligand IgA) and CD89.
[283] Mab 14.1 did not inhibit IgA and MY43 binding, but inhibited the binding of A77 and A59 with CD89, indicating that the antibody binds at a position other than the natural ligand binding site.
[284] Mab 7.4 did not inhibit the binding of IgA, MY43, A77 or A59 to CD89, indicating that the antibody also binds at a position other than the natural ligand binding site but at a site other than 14.1.
[285] Blocking studies
[286] 25 μl of antibody dilution was added to 50,000 2a1.6 / CD89 in 50 μl of Facs buffer and incubated at 4 ° C. for 15 minutes. Suboptimal concentrations of 25 μl of A77-FITC, A59-PE or MY43 were added and incubated at 4 ° C. for 30 minutes and washed three times with 100 μl of Facs buffer. Cells treated with MY43 were incubated with rabbit (Fab ₂ ) anti-Hu IgG-FITC for 30 minutes at 4 ° C. and washed three times with 100 μl of Facs buffer. Cells were then resuspended in 200 μl of Facs buffer. Fluorescence was measured on a faxcaliber (Becton Dickinson).
[287] Inhibition ELISA
[288] 96-well ELISA (flat) bottom plates were coated using 100 μl / well of soluble recombinant CD89 (1 μg / ml) in PBS and incubated overnight at room temperature. ELISA plates were shaken off and incubated with 100 μl PBS / 0.05% Tween / 1% Chicken Serum (PBST / Ch) per well for 60 minutes at room temperature. Plates were shaken off and incubated for 10 minutes with excess antibody (50 μl / well) diluted in PBST / Ch. The aIgA2 solution was added in a dose response of 50 μl per well in PBST / Ch and incubated for 60 minutes at room temperature. Plates were then washed three times with PBS / 0.05% Tween (PBST) and incubated for 60 minutes at room temperature with 100 μl of 4E8-Biotin (mouse anti-IgA) per well. Plates were then washed three times with PBST, incubated with 100 μl Strep-HRP in PBST / Ch per well for 60 minutes at room temperature, washed three times with PBST to perform ELISA with ABTS. The optimal density at a wavelength of 405 nm was read in a Bio-tek reader.
[289] III. Immunoprecipitation Research
[290] Mab 14.1 and 7.4 were found to immunoprecipitate soluble recombinant CD89 (sCD89).
[291] Briefly, the ProtG Sepharose Fast Flow column was washed three times with PBS. SCD89 was preliminarily removed at 4 ° C. for 2 hours with washed beads in PBS. Pre-depleted sCD89 was incubated with mouse IgG or human IgG antibody at 4 ° C. for 2 hours. The CD89 / IgG solution was washed with ProtG at 4 ° C. for 1 hour and then 5 times with PBS. The samples were then resuspended in SDS-PAGE sample buffer and separated on SDS-PAGE gradient gels before staining the gels with Comassie Brilliant Blue.
[292] IV. Affinity research
[293] The affinity kinetics of MAbs 14.1 and 7.4, ie the binding equilibrium binding constants (K _a ) and dissociation constants (K _d ), were determined as follows:
[294] For Mab 14.1, K _a was measured to be about 1.5 × 10 ⁹ and K _d to be about 6.8 × 10 ⁻¹⁰ .
[295] For MAb 7.4, K _a was determined to be about 2.0 × 10 ⁸ and K _d was about 5.1 × 10 ⁻⁹ .
[296] Affinity reaction rate was measured using BIAcore analysis by immobilizing sCD89 on the chip and flowing various concentrations of Mab on the chip. Mab capture analysis was also performed by flowing sCD89 into MAb. Immobilization of the antigen and human IgG was performed via amine coupling. The kit was purchased from BIAcore, which contains chips and buffers. It was constructed using the langmuir model. Data analysis was performed through a model available in the software associated with the device.
[297] Example 5: Activity of Human Monoclonal Antibodies Against CD89
[298] I. Calcium Flux Studies
[299] Mab 14.1 was tested as follows to find that it induced calcium efflux. Mab 7.4 did not induce calcium efflux.
[300] After 3 × 10 ⁶ cells were resuspended in 1.52 ml of serum-free medium and washed three times, 38 μl SNARF-1 and 38 μl FLUO-3 were added. Cells were incubated at 37 ° C. under dark conditions and 5 ml of warm (37 ° C.) serum free medium was added, and cells were incubated at 37 ° C. for 5 minutes. Cells were then washed twice with serum free medium and again washed with coating with antibody anti-FcαR1. Cells were resuspended in Ca ⁺⁺ Mobilisation buffer and run for 24 seconds with a flow cytometer to establish baseline levels. Cross-linked antibody anti-Hu IgG-Fc cells were then added and monitored for 4 minutes. To establish a negative control, cells that were not incubated with anti-FcocR1 were used.
[301] II. Human C1q Binding Study
[302] Using ELISA and FACS as described in the following paragraphs, Mab 14.1 and 7.4 were tested for fixation of hC1q (ELISA) and even when bound to CD89 expressing cells (FACS analysis).
[303] Mab 14.1 was able to fix hC1q in ELISA and weakly when bound to cells. Mab 7.4 was able to fix hC1q in ELISA but not when bound to cells. When bound to cells, it reflects in vivo conditions, where the inability of these Mabs to immobilize hC1q is their use in therapy, eg in situations where it is undesirable to initiate a complement cascade (target cell lysis occurs). To make it especially valuable.
[304] ELISA analysis
[305] Briefly, 96-well ELISA (flat) bottom plates were incubated overnight at room temperature using 100 μl / well of human IgG (3 μg / ml) in PBS (using hIgG1, hIgG2, hIgG3 and hIGg4 as controls). After shaking off, 100 μl of phosphate / NaCl / gelatin / twin buffer (C1q-ELISA dilution) per well was coated for 60 minutes at room temperature. The plates were shaken off and diluted in C1a-ELISA dilutions and incubated at 20 μg / ml (100 μl / well) for 60 minutes at room temperature and washed three times with C1q-ELISA dilutions. Next, rabbit anti-hC1q diluted in 100 μl of C1q-ELISA dilution per well was added to the plate and incubated at room temperature for 60 minutes, washed three times with C1q-ELISA dilution to 100 μl of C1q-ELISA dilution per well. Incubated with PO-conjugated porcine anti-Rb IgG-Fc diluted for 60 min at room temperature. The plates were then washed three times with C1q-ELISA dilutions. ELISA was developed with ABTS and the optimal density was read at a wavelength of 405 nm in a bio-tech reader.
[306] FACS analysis
[307] 25 μl of antibody dilution in C1q-Facs buffer was added to 50,000 2a1.6 / CD89 cells in 50 μl of Facs buffer and NaCl (C1q-Facs buffer) and incubated for 15 minutes at 4 ° C., where C1q-Facs buffer was added. 25 μl of hC1q (20 μg / ml) was added and incubated at 4 ° C. for 30 minutes. Cells were then washed three times with 100 μl C1q-Facs buffer and incubated with rabbit anti-hC1q-FITC for 30 minutes at 4 ° C. and washed three times with 100 μl of C1q-Facs buffer. Cells were resuspended in 200 μl C1q-Facs buffer. Fluorescence was measured on a faxcaliber (Becton Dickinson).
[308] Equivalent
[309] Those skilled in the art will be able to recognize or identify many equivalents of the specific embodiments of the invention described herein without using experiments other than routine experiments. Such equivalents are included in the following claims.
SEQUENCE LISTING<110> Medarex, Inc. et al.<120> HUMAN MONOCLONAL ANTIBODIES TO FC ALPHA      RECEPTOR (CD89)<130> MXI-211<150> US 60 / 338,956<151> 2001-11-05<150> US 60 / 268,075<151> 2001-02-12<160> 4FastSEQ for Windows Version 4.0<210> 1<211> 357<212> DNA<213> Homo sapiens<400> 1caggtgcaac tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agttatgttc tgcactgggt ccgccaggct 120ccaggcaagg ggctggattg ggtggcagtg atatcagatg atggaaggaa taaatacttc 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gagagaaggg 300tatagcggca gctggtttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357<210> 2<211> 119<212> PRT<213> Homo sapiens<400> 2Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr            20 25 30Val Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val        35 40 45Ala Val Ile Ser Asp Asp Gly Arg Asn Lys Tyr Phe Ala Asp Ser Val    50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                85 90 95Val Arg Glu Gly Tyr Ser Gly Ser Trp Phe Asp Tyr Trp Gly Gln Gly            100 105 110Thr Leu Val Thr Val Ser Ser        115<210> 3<211> 321<212> DNA<213> Homo sapiens<400> 3gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca 120gggaaagctc ctaagctcct gatctatggt gcctccagtt tggaaggtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag tttaatagtt acccattcac tttcggccct 300gggaccaaag tggatatcaa a 321<210> 4<211> 107<212> PRT<213> Homo sapiens<400> 4Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala            20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile        35 40 45Tyr Gly Ala Ser Ser Leu Glu Gly Gly Val Pro Ser Arg Phe Ser Gly    50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Phe                85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys            100 105<210> 5<211> 357<212> DNA<213> Homo sapiens<400> 5caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagaaa taaagactac 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgc attactgtgc gaggcttgac 300tggggatatg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcttca 357<210> 6<211> 119<212> PRT<213> Homo sapiens<400> 6Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr            20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val        35 40 45Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Asp Tyr Ala Asp Ser Val    50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val His Tyr Cys                85 90 95Ala Arg Leu Asp Trp Gly Tyr Asp Ala Phe Asp Ile Trp Gly Gln Gly            100 105 110Thr Met Val Thr Val Ser Ser        115<210> 7<211> 327<212> DNA<213> Homo sapiens<400> 7gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaag 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcc gtacactttt 300ggccagggga ccaagctgga gatcaaa 327<210> 8<211> 109<212> PRT<213> Homo sapiens<400> 8Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser            20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu        35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser    50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                85 90 95Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys            100 105

权利要求:
Claims (51)
[1" claim-type="Currently amended] Isolated human monoclonal antibody that binds human CD89.
[2" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein said antibody does not activate complement.
[3" claim-type="Currently amended] The isolated antibody of claim 1, wherein said antibody binds to a CD89 site that is different from the IgA binding site of the CD89 receptor.
[4" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein the antibody inhibits binding of IgA to CD89.
[5" claim-type="Currently amended] The isolated human monoclonal antibody of claim 4, wherein the antibody inhibits at least about 50% binding of IgA to CD89.
[6" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein the antibody binds to human CD89 with an equilibrium binding constant (K _a ) of at least 10 ⁸ M ⁻¹ .
[7" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein the antibody binds to human CD89 with an equilibrium binding constant (K _a ) of at least 10 ⁹ M ⁻¹ .
[8" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein the antibody heavy chain is an IgG1 heavy chain and the antibody light chain is a kappa light chain.
[9" claim-type="Currently amended] The light chain encoded by a nucleic acid comprising a heavy chain encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 5, a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7, And isolated human monoclonal antibodies comprising conservative sequence variants thereof.
[10" claim-type="Currently amended] The method of claim 1, comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6, a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, and conservative sequence variants thereof. Isolated human monoclonal antibody.
[11" claim-type="Currently amended] (a) the binding equilibrium binding constant (K _a ) for human CD89 is at least about 10 ⁷ M ⁻¹ ;
(b) the dissociation constant (K _d ) from human CD89 is about 10 ⁻⁸ S ⁻¹ or less;
(c) not activating complement upon binding to human CD89 in vivo;
(d) the antibody binds to an epitope on human CD89 without inhibiting human IgA binding to the human CD89 receptor;
(e) the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6 and conservative sequence variants thereof, and an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8 and conservative sequence variants thereof Involving light chains
An isolated human monoclonal antibody that binds to human CD89 having at least one feature selected from the group consisting of:
[12" claim-type="Currently amended] The isolated human monoclonal antibody of claim 1, wherein the antibody is a Fab fragment or a single chain antibody.
[13" claim-type="Currently amended] The antibody of claim 1, wherein the antibody comprises B cells obtained from a transgenic animal other than human having a genome comprising a human heavy chain transgene and a human light chain transgene and fused with endogenous proliferating cells. Isolated human monoclonal antibody produced by hybridoma.
[14" claim-type="Currently amended] A B cell obtained from a non-human transgenic animal having a genome comprising a human heavy chain transgene and a human light chain transgene and fused with an infinite proliferation cell, wherein the human monoclonal antibody according to claim 1 is detected in a detectable amount. Hybridoma to produce.
[15" claim-type="Currently amended] 15. The hybridoma of claim 14, wherein the hybridoma is selected from the group consisting of hybridomas designated 7.4, 8.2, and 14.1.
[16" claim-type="Currently amended] Isolated human monoclonal antibody that binds to an epitope on human CD89 defined by an antibody selected from the group consisting of mAbs 7.4, 8.2 and 14.1.
[17" claim-type="Currently amended] An isolated human monoclonal antibody that binds to an epitope on human CD89 defined by mAb 14.1 having a heavy chain amino acid sequence as shown in SEQ ID NO: 2 and a light chain amino acid sequence as shown in SEQ ID NO: 4.
[18" claim-type="Currently amended] Isolated human monoclonal antibody having the binding properties of the antibody selected from the group consisting of mAbs 7.4, 8.2 and 14.1.
[19" claim-type="Currently amended] An isolated human monoclonal antibody having the binding properties of mAb 14.1 having the heavy chain amino acid sequence shown in SEQ ID NO: 2 and the light chain amino acid sequence shown in SEQ ID NO: 4.
[20" claim-type="Currently amended] A transgenic animal other than human expressing the antibody of claim 1 having a genome comprising a human heavy chain transgene and a human light chain transgene.
[21" claim-type="Currently amended] Immunizing a non-human transgenic animal having a genome comprising a human heavy chain transgene and a human light chain transgene with cells expressing CD89 or CD89 such that antibodies are produced by the B cells of the animal;
Isolating B cells of said animal; And
Fusing the B cells with myeloma cells to form an infinite proliferation hybridoma cell that secretes the antibody
Including, the method of producing the antibody of claim 1.
[22" claim-type="Currently amended] A bispecific or multispecific molecule comprising the human antibody of claim 16 and a moiety that binds to a target antigen other than CD89.
[23" claim-type="Currently amended] The bispecific or multispecific molecule of claim 22, wherein said antibody is a Fab fragment or a single chain antibody.
[24" claim-type="Currently amended] The bispecific or multispecific molecule of claim 22, wherein said target antigen is a tumor antigen.
[25" claim-type="Currently amended] The bispecific or multispecific molecule of claim 22, wherein the moiety that binds to the target antigen comprises an antibody or tumor ligand.
[26" claim-type="Currently amended] The bispecific or multispecific molecule of claim 22, comprising the fusion protein.
[27" claim-type="Currently amended] 23. The bispecific or multispecific molecule of claim 22 comprising a chemically linked conjugate.
[28" claim-type="Currently amended] The bispecific or multispecific molecule of claim 22 which induces lysis (ADCC) of cells expressing the target antigen in the presence of effector cells expressing CD89.
[29" claim-type="Currently amended] 23. The method of claim 22, wherein said antigen is a carcinoblastic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER2 / neu , HER3, HER4 , Bispecific or multispecific molecule selected from the group consisting of: CD20, CD30, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, and TAG 72.
[30" claim-type="Currently amended] A molecular conjugate comprising the human antibody of claim 16 linked to an antigen.
[31" claim-type="Currently amended] The molecular conjugate of claim 30, wherein said antibody is a Fab fragment or a single chain antibody.
[32" claim-type="Currently amended] 31. The molecular conjugate of claim 30, comprising a fusion protein.
[33" claim-type="Currently amended] 31. The molecular conjugate of claim 30, wherein the molecular conjugate comprises a chemically linked conjugate.
[34" claim-type="Currently amended] The molecular conjugate of claim 30, wherein said antigen is a tumor antigen.
[35" claim-type="Currently amended] A composition comprising the human antibody of claim 1 and a pharmaceutically acceptable carrier.
[36" claim-type="Currently amended] A composition comprising the bispecific molecule of claim 22 and a pharmaceutically acceptable carrier.
[37" claim-type="Currently amended] A composition comprising the molecular conjugate of claim 30 and a pharmaceutically acceptable carrier.
[38" claim-type="Currently amended] A composition comprising a combination of two or more human antibodies of claim 1, wherein each of these antibodies binds to a separate epitope on human CD89.
[39" claim-type="Currently amended] 36. The composition of claim 35, further comprising a cytotoxic agent.
[40" claim-type="Currently amended] Immunotoxin comprising the antibody of claim 2 linked to a cytotoxic agent.
[41" claim-type="Currently amended] A method of inhibiting cell growth comprising contacting a cell with an effective amount of the bispecific antibody of claim 22 comprising a moiety that binds an antigen on the cell.
[42" claim-type="Currently amended] 42. The method of claim 41, wherein said antigen is a tumor antigen.
[43" claim-type="Currently amended] The method of claim 42, wherein the tumor antigen is from a cancer cell selected from the group of cancers consisting of ovarian cancer, breast cancer, testicular cancer, prostate cancer, leukemia and lymphoma.
[44" claim-type="Currently amended] 42. The method of claim 41, wherein said antigen is an autoantigen.
[45" claim-type="Currently amended] 42. The method of claim 41 wherein the antigen is from a microorganism.
[46" claim-type="Currently amended] 46. The method of claim 45, wherein said microorganism is selected from the group consisting of bacteria, viruses and parasites.
[47" claim-type="Currently amended] In a subject in need of treatment of a disease characterized by precipitation of an IgA-immune complex, an isolated human monoclonal antibody that specifically binds to CD89 and blocks IgA from binding to CD89 is treated or prevented. A method for treating or preventing a disease characterized by the precipitation of an IgA-immune complex, comprising administering in an amount effective to a.
[48" claim-type="Currently amended] 48. The method of claim 47, wherein said disease characterized by the precipitation of IgA-immune complexes is chronic hepatitis, Henoch-Schonlein purpura (HSP), Burger's disease, and IgA-glomerulonephritis. And selected from the group consisting of:
[49" claim-type="Currently amended] The method of claim 47, wherein said monoclonal antibody binds to an epitope on CD89 defined by antibody 8.2.
[50" claim-type="Currently amended] Contacting the sample with the antibody of claim 1 under conditions such that the antibody and CD89 can form a complex; And
Detecting the formation of the complex
A method for detecting the presence of CD89 or the presence of cells expressing CD89 in a sample, comprising.
[51" claim-type="Currently amended] A nucleotide sequence encoding the variable and constant regions of the heavy and light chains of a human monoclonal antibody that binds to human CD89, wherein the heavy chain nucleotide sequence is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 5, and the light chain nucleotide sequence is An expression vector selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7.

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同族专利:

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公开号 | 公开日

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AU2002240338B2|2005-10-27|

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JP2010004888A|2010-01-14|

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MXPA03007144A|2004-04-02|

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WO2002064634A3|2003-09-25|

---

ZA200305996B|2005-06-29|

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EP1370588A2|2003-12-17|

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US7192582B2|2007-03-20|

---

IL157274A|2010-04-15|

---

CA2437814A1|2002-08-22|

---

AU2002240338C1|2007-05-17|

---

CA2437814C|2008-05-13|

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NZ527977A|2005-10-28|

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AU2002240338B9|2006-02-23|

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CN1312181C|2007-04-25|

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KR100890088B1|2009-03-24|

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HK1065051A1|2007-10-18|

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JP2005507635A|2005-03-24|

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US20030082643A1|2003-05-01|

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WO2002064634A2|2002-08-22|

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IL157274D0|2004-02-19|

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CN1500097A|2004-05-26|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-02-12|Priority to US26807501P

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2001-02-12|Priority to US60/268,075

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2001-11-05|Priority to US33895601P

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2001-11-05|Priority to US60/338,956

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2002-02-11|Application filed by 메다렉스, 인코포레이티드

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2002-02-11|Priority to PCT/US2002/004024

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2004-07-09|Publication of KR20040062873A

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2009-03-24|Application granted

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2009-03-24|Publication of KR100890088B1

优先权:

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申请号 | 申请日 | 专利标题

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US26807501P| true| 2001-02-12|2001-02-12|

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US60/268,075|2001-02-12|

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US33895601P| true| 2001-11-05|2001-11-05|

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US60/338,956|2001-11-05|

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PCT/US2002/004024|WO2002064634A2|2001-02-12|2002-02-11|Human monoclonal antibodies to fc alpha receptor |