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Binding domain-immunoglobulin fusion proteins

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US20030133939A1
US20030133939A1 US10053530 US5353002A US2003133939A1 US 20030133939 A1 US20030133939 A1 US 20030133939A1 US 10053530 US10053530 US 10053530 US 5353002 A US5353002 A US 5353002A US 2003133939 A1 US2003133939 A1 US 2003133939A1
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Jeffrey Ledbetter
Martha Hayden-Ledbetter
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Emergent Product Development Seattle LLC
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Genecraft Inc
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    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46Hybrid immunoglobulins
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07K16/46Hybrid immunoglobulins
    • C07K16/461Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/462Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
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    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00Immunoglobulins specific features
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    • C07K2317/52Constant or Fc region; Isotype
    • C07K2317/53Hinge
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734Complement-dependent cytotoxicity [CDC]
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    • C07K2319/00Fusion polypeptide
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    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a hinge region polypeptide having either zero or one cysteine residue, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and/or CDC while occurring predominantly as monomeric polypeptides. The fusion proteins can be recombinantly produced at high expression levels. Also provided are related compositions and methods, including immunotherapeutic applications.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • [0001]
    This application claims the benefit of priority of U.S. Provisional Application No. 60/______ (to be assigned, formerly U.S. application Ser. No. 09/765,208, filed Jan. 17, 2001). A Petition to Convert a Non-Provisional Application to a Provisional Application was filed on Jan. 16, 2002 (copy attached).
  • BACKGROUND OF THE INVENTION
  • [0002]
    The present invention relates generally to immunologically active, recombinant binding proteins, and in particular, to molecularly engineered binding domain-immunoglobulin fusion proteins, including single chain Fv-immunoglobulin fusion proteins. The present invention also relates to compositions and methods for treating malignant conditions and B-cell disorders, including diseases characterized by autoantibody production.
  • [0003]
    An immunoglobulin molecule is composed of two identical light chains and two identical heavy chains that are joined into a macromolecular complex by interchain disulfide bonds. Intrachain disulfide bonds join different areas of the same polypeptide chain, which results in the formation of loops that along with adjacent amino acids constitute the immunoglobulin domains. Each light chain and each heavy chain has a single variable region that shows considerable variation in amino acid composition from one antibody to another. The light chain variable region, VL, associates with the variable region of a heavy chain, VH, to form the antigen binding site of the immunoglobulin, Fv Light chains have a single constant region domain and heavy chains have several constant region domains. Classes IgG, IgA and IgE have three constant region domains, which are designated CH1, CH2, and CH3, and the IgM and IgE classes have four constant region domains.
  • [0004]
    The heavy chains of immunoglobulins can be divided into three functional regions: Fd, hinge, and Fc. The Fd region comprises the VH and CH1 domains and in combination with the light chain forms Fab. The Fc fragment is generally considered responsible for the effector functions of an immunoglobulin, such as, complement fixation and binding to Fc receptors. The hinge region, found in IgG, IgA, and IgD classes, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, three human IgG subclasses, IgG1, IgG2, and IgG4, have hinge regions of 12-15 amino acids while IgG3 comprises approximately 62 amino acids, including 21 proline residues and 11 cysteine residues. According to crystallographic studies, the hinge can be further subdivided functionally into three regions: the upper hinge, the core, and the lower hinge (Shin et al., Immunological Reviews 130:87 (1992)). The upper hinge includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. (Id.) The core hinge region of human IgG1 contains the sequence, Cys-Pro-Pro-Cys, which when disulfide bonds are formed results in a cyclic octa-peptide believed to act as a pivot, thus conferring flexibility. The hinge region may also contain carbohydrate attachment sites. For example, IgA1 contains five carbohydrate sites within a 17 amino acid segment of the hinge region, conferring exception resistance of the hinge to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
  • [0005]
    Conformational changes permitted by the structure and flexibility of the hinge region may affect the effector functions of the Fc portion of the antibody. Three general categories of effector functions associated with the Fc region include (1) activation of the classical complement cascade, (2) interaction with effector cells, and (3) compartmentalization of immunoglobulins. The different human IgG subclasses vary in their relative efficacy to activate and amplify the steps of the complement cascade. In general, IgG1 and IgG3 most effectively fix complement, IgG2 is less effective, and IgG4 does not activate complement. Complement activation is initiated by binding of C1q , a subunit of the first component C1 in the cascade, to an antigen-antibody complex. Even though the binding site for C1q is located in the CH2 domain of the antibody, the hinge region influences the ability of the antibody to activate the cascade. For example, recombinant immunoglobulins lacking a hinge region are unable to activate complement. (Id.) Without the flexibility conferred by the hinge region, the Fab portion of the antibody bound to the antigen may not be able to adopt the conformation required to permit C1q to bind to CH2. (See id.) Studies have indicated that hinge length and segmental flexibility correlate with complement activation; however, the correlation is not absolute. Human IgG3 molecules with altered hinge regions that are as rigid as IgG4 still effectively activate the cascade.
  • [0006]
    Lack of the hinge region also affects the ability of human IgG immunoglobulins to bind Fc receptors on immune effector cells. Binding of an immunoglobulin to an Fc receptor facilitates antibody-dependent cellular cytotoxicity (ADCC), which is presumed to be an important means to eliminate tumor cells. The human IgG Fc receptor family is divided into three groups, FcγRI (CD64), which is capable of binding IgG with high affinity, FcγRII (CD32), and FcγRIII (CD16), both of which are low affinity receptors. The molecular interaction between each of the three receptors and an immunoglobulin has not been defined precisely, but experiments indicate that residues in the hinge proximal region of the CH2 domain are important to the specificity of the interaction between the antibody and the Fc receptor. In addition, IgG 1 myeloma proteins and recombinant IgG3 chimeric antibodies that lack a hinge region are unable to bind FcγRI, likely because accessibility to CH2 is decreased. (Shin et al., Intern Rev, Immunol. 10: 177, 178-79 (1993))
  • [0007]
    Monoclonal antibody technology and genetic engineering methods have led to rapid development of immunoglobulin molecules for diagnosis and treatment of human diseases. Protein engineering has been applied to improve the affinity of an antibody for its cognate antigen, to diminish problems related to immunogenicity, and to alter an antibody's effector functions. The domain structure of immunoglobulins is amenable to engineering, in that the antigen binding domains and the domains conferring effector functions may be exchanged between immunoglobulin classes and subclasses.
  • [0008]
    In addition, smaller immunoglobulin molecules have been constructed to overcome problems associated with whole immunoglobulin therapy. Single chain Fv (scFv) comprise the heavy chain variable domain joined via a short linker peptide to the light chain variable domain (Huston et al. Proc. Natl. Acad. Sci. USA, 85: 5879-83, 988). Because of the small size of scFv molecules, they exhibit very rapid clearance from plasma and tissues and more effective penetration into tissues than whole immunoglobulin. An anti-tumor scFv showed more rapid tumor penetration and more even distribution through the tumor mass than the corresponding chimeric antibody (Yokota et al., Cancer Res 52, 3402-08 (1992)). Fusion of an scFv to another molecule, such as a toxin, takes advantage of the specific antigen-binding activity and the small size of an scFv to deliver the toxin to a target tissue. (Chaudary et al., Nature 339:394 (1989); Batra et al., Mol. Cell. Biol. 11:2200 (1991)).
  • [0009]
    Despite the advantages that scFv molecules bring to serotherapy, several drawbacks to this therapeutic approach exist. While rapid clearance of scFv may reduce toxic effects in normal cells, such rapid clearance may prevent delivery of a minimum effective dose to the target tissue. Manufacturing adequate amounts of scFv for administration to patients has been challenging due to difficulties in expression and isolation of scFv that adversely affect the yield. During expression, scFv molecules lack stability and often aggregate due to pairing of variable regions from different molecules. Furthermore, production levels of scFv molecules in mammalian expression systems are low, limiting the potential for efficient manufacturing of scFv molecules for therapy (Davis et al, J Biol. Chem. 265:10410-18 (1990); Traunecker et al., EMBO J 10: 3655-59 (1991)). Strategies for improving production have been explored, including addition of glycosylation sites to the variable regions (Jost, C. R. U.S. Pat. No. 5,888,773, Jost et al, J. Biol. Chem. 269: 26267-73 (1994)).
  • [0010]
    Conjugation or fusion of toxins to scFV provides a very potent molecule, but dosing is limited by toxicity from the toxin molecule. Toxic effects include elevation of liver enzymes and vascular leak syndrome. In addition, immunotoxins are highly immunogenic, and host antibodies generated against the toxin limit its potential for repeated treatment.
  • [0011]
    An additional disadvantage to using scFv for therapy is the lack of effector function. An scFv without the cytolytic functions, ADCC and complement dependent-cytotoxicity (CDC), associated with the constant region of an immunoglobulin may be ineffective for treating disease. Even though development of scFv technology began over 12 years ago, currently no scFv products are approved for therapy.
  • [0012]
    The benefit of antibody constant region-associated effector functions to treatment of a disease has prompted development of fusion proteins in which nonimmunoglobulin sequences are substituted for the antibody variable region. For example, CD4, the T cell surface protein recognized by HIV, was recombinantly fused to an immunoglobulin Fc effector domain. (See Sensel et al., Chem Immunol. 65:129-158 (1997)). The biological activity of such a molecule will depend in part on the class or subclass of the constant region chosen. An IL-2-IgG1 fusion protein effected complement-mediated lysis of IL-2 receptor-bearing cells. (See id.). Use of immunoglobulin constant regions to construct these and other fusion proteins may also confer improved pharmacokinetic properties.
  • [0013]
    Diseases and disorders thought to be amenable to some type of immunoglobulin therapy include cancer and immune system disorders. Cancer includes a broad range of diseases, affecting approximately one in four individuals worldwide. Rapid and unregulated proliferation of malignant cells is a hallmark of many types of cancer, including hematological malignancies. Patients with a hematologic malignant condition have benefited most from advances in cancer therapy in the past two decades (Multani et al., J. Clin. Oncology 16: 3691-3710, 1998). Although remission rates have increased, most patients still relapse and succumb to their disease. Barriers to cure with cytotoxic drugs include tumor cell resistance and the high toxicity of chemotherapy, which prevents optimal dosing in many patients. New treatments based on targeting with molecules that specifically bind to a malignant cell, including monoclonal antibodies (mAbs), can improve effectiveness without increasing toxicity.
  • [0014]
    Since mAbs were first described in 1975 (Kohler et al., Nature 256:495-97 (1975)), many patients have been treated with mAbs to antigens expressed on tumor cells. These studies have yielded important lessons regarding the selection of target antigens suitable for therapy. First and most importantly, the target antigen should not be expressed by crucial normal tissues. Fortunately, hematologic malignant cells express many antigens that are not expressed on stem cells or other essential cells. Treatment of a hematologic malignant condition that depletes both normal and malignant cells of hematological origin has been acceptable because regeneration of normal cells from progenitors occurs after therapy has ended. Second, the target antigen should be expressed on all clonogenic populations of tumor cells, and expression should persist despite the selective pressure from immunoglobulin therapy. Thus, the choice of surface idiotype for therapy of B cell malignancy has been limited by the outgrowth of tumor cell variants with altered surface idiotype expression even though the antigen exhibits a high degree of tumor selectivity (Meeker et al., N. Engl. J. Med. 312:1658-65 (1985)). Third, the selected antigen must traffic properly after an immunoglobulin binds to it. Shedding or internalization of a target antigen after an immunoglobulin binds to the antigen may allow tumor cells to escape destruction, thus limiting the effectiveness of serotherapy. Fourth, binding of an immunoglobulin to target antigens that transmit activation signals may result in improved functional responses in tumor cells that lead to growth arrest and apoptosis. While all of these properties are important, the triggering of apoptosis after an immunoglobulin binds to the antigen may be a critical factor in achieving successful serotherapy.
  • [0015]
    Antigens that have been tested as targets for serotherapy of B and T cell malignancies include Ig idiotype (Brown et al., Blood 73:651-61 (1989)), CD19 (Hekman et al., Cancer Immunol. Immunother. 32:364-72 (1991); Vlasveld et al., Cancer Immunol. Immunother. 40: 37-47 (1995)), CD20 (Press et al., Blood 69: 584-91 (1987); Maloney et al., J. Clin. Oncol. 15:3266-74, (1997)) CD21 (Scheinberg et. al., J. Clin. Oncol. 8:792-803, (1990)), CD5 (Dillman et. al., J. Biol. Respn. Mod. 5:394-410 (1986)), and CD52 (CAMPATH) (Pawson et al, J. Clin. Oncol. 15:2667-72, (1997)). Of these, the most success has been obtained using CD20 as a target for therapy of B cell lymphomas. Each of the other targets has been limited by the biological properties of the antigen. For example, surface idiotype can be altered through somatic mutation, allowing tumor cell escape. CD5, CD21, and CD19 are rapidly internalized after mAb binding, allowing tumor cells to escape destruction unless mAbs are conjugated with toxin molecules. CD22 is expressed on only a subset of B cell lymphomas, while CD52 is expressed on both T cells and B cells and generates immunosuppression from T cell depletion.
  • [0016]
    CD20 fulfills the basic criteria described above for selection of an appropriate target antigen for therapy of a B cell malignant condition. Treatment of patients with low grade or follicular B cell lymphoma using chimeric CD20 mAb induces partial or complete responses in many patients (McLaughlin et al, Blood 88:90a (abstract, suppl. 1) (1996); Maloney et al, Blood 90: 2188-95 (1997)). However, tumor relapse commonly occurs within six months to one year. Therefore, further improvements in serotherapy are needed to induce more durable responses in low grade B cell lymphoma, and to allow effective treatment of high grade lymphoma and other B cell diseases.
  • [0017]
    One approach to improving CD20 serotherapy has been to target radioisotopes to B cell lymphomas using mAbs specific for CD20. While the effectiveness of therapy is increased, associated toxicity from the long in vivo half-life of the radioactive antibody increased also, sometimes requiring that the patient undergo stem cell rescue (Press et al., N. Eng. J. Med. 329: 1219-1224, 1993; Kaminski et al., N. Eng. J. Med. 329:459-65 (1993)). MAbs to CD20 have been cleaved with proteases to yield F(ab′)2 or Fab fragments prior to attachment of the radioisotope. This improves penetration of the radioisotope conjugate into the tumor, and shortens the in vivo half-life, thus reducing the toxicity to normal tissues. However, the advantages of effector functions, including complement fixation and ADCC, that are provided by the Fc region of the CD20 mAb are lost. Therefore, for improved delivery of radioisotopes, a strategy is needed to make a CD20 mAb derivative that retains Fc-dependent effector functions but is smaller in size, thereby increasing tumor penetration and shortening mAb half-life.
  • [0018]
    CD20 was the first human B cell lineage-specific surface molecule identified by a monoclonal antibody, but the function of CD20 in B cell biology is still incompletely understood. CD20 is a non-glycosylated, hydrophobic 35 kDa phosphoprotein that has both amino and carboxy ends in the cytoplasm (Einfeld et al, EMBO J. 7:711-17 (1988)). Natural ligands for CD20 have not been identified. CD20 is expressed by all normal mature B cells, but is not expressed by precursor B cells.
  • [0019]
    CD20 mAbs deliver signals to normal B cells that affect viability and growth (Clark et al., Proc. Natl. Acad Sci. USA 83:4494-98 (1986)). Recent data has shown that extensive cross-linking of CD20 can induce apoptosis of B lymphoma cell lines (Shan et al., Blood 91:1644-52 (1998)). Cross-linking of CD20 on the cell surface increases the magnitude and kinetics of signal transduction, which was detected by measuring phosphorylation of cellular substrates on tyrosine residues (Deans et al., J. Immunol. 146:846-53 (1993)). Importantly, apoptosis of Ramos B lymphoma cells was also be induced by cross-linking of CD20 mAbs by addition of Fc-receptor positive cells (Shan et al., Blood 91: 1644-52 (1998)). Therefore, in addition to cellular depletion by complement and ADCC mechanisms, Fc-receptor binding by CD20 mAbs in vivo could promote apoptosis of malignant B cells by CD20 cross-linking. This theory is consistent with experiments showing that effectiveness of CD20 therapy of human lymphoma in a SCID mouse model was dependent upon Fc-receptor binding by the CD20 mAb (Funakoshi et al., J. Immunotherapy 19:93-101 (1996))
  • [0020]
    The CD20 polypeptide contains four transmembrane domains (Einfeld et al., EMBO J. 7: 711-17, (1988); Stamenkovic et al., J. Exp. Med. 167;1975-80 (1988); Tedder et. al., J. Immunol. 141:4388-4394 (1988)). The multiple membrane spanning domains prevent CD20 internalization after antibody binding. This property of CD20 was recognized as an important feature for effective therapy of B cell malignancies when a murine CD20 mAb, 1F5, was injected into patients with B cell lymphoma, resulting in significant depletion of malignant cells and partial clinical responses (Press et al., Blood 69: 584-91 (1987)).
  • [0021]
    Because normal mature B cells also express CD20, normal B cells are depleted during CD20 antibody therapy (Reff, M. E. et al, Blood 83: 435-445, 1994). However, after treatment is completed, normal B cells are regenerated from CD20 negative B cell precursors; therefore, patients treated with anti-CD20 therapy do not experience significant immunosuppression. Depletion of normal B cells may be beneficial in diseases that involve inappropriate production of autoantibodies or other diseases where B cells may play a role. A chimeric mAb specific for CD20, consisting of heavy and light chain variable regions of mouse origin fused to human IgG1 heavy chain and human kappa light chain constant regions, retained binding to CD20 and the ability to mediate ADCC and to fix complement (Liu et al., J. Immunol. 139:3521-26 (1987); Robinson et al., U.S. Pat. No. 5,500,362). This work led to development of a chimeric CD20 mAb, Rituximab™, currently approved by the U.S. Food and Drug Administration for approval for therapy of B cell lymphomas. While clinical responses are frequently observed after treatment with Rituximab, patients often relapse after about 6-12 months.
  • [0022]
    High doses of Rituximab™ are required for intravenous injection because the molecule is large, approximately 150 kDa, and diffusion is limited into the lymphoid tissues where many tumor cells reside. The mechanism of anti-tumor activity of Rituximab™ is thought to be a combination of several activities, including ADCC, fixation of complement, and triggering of signals in malignant B cells that promote apoptosis. The large size of Rituximab™ prevents optimal diffusion of the molecule into lymphoid tissues that contain malignant B cells, thereby limiting these anti-tumor activities. As discussed above, cleavage of CD20 mAbs with proteases into Fab or F(ab′)2 fragments makes them smaller and allows better penetration into lymphoid tissues, but the effector functions important for anti-tumor activity are lost. While CD20 mAb fragments may be more effective than intact antibody for delivery of radioisotopes, it would be desirable to construct a CD20 mAb derivative that retains the effector functions of the Fc portion, but is smaller in size, facilitating better tumor penetration and resulting in a shorter half-life.
  • [0023]
    CD20 is expressed by malignant cells of B cell origin, including B cell lymphoma and chronic lymphocytic leukemia (CLL). CD20 is not expressed by malignancies of pre-B cells, such as acute lymphoblastic leukemia. CD20 is therefore a good target for therapy of B cell lymphoma, CLL, and other diseases in which B cells are involved in the disease activity. Other B cell disorders include autoimmune diseases in which autoantibodies are produced during the differentiation of B cells into plasma cells. Examples of B cell disorders include autoimmune thyroid disease, including Graves'disease and Hashimoto's thyroiditis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogrens syndrome, immune thrombocytopenic purpura (ITP), multiple sclerosis (MS), myasthenia gravis (MG), psoriasis, scleroderma, and inflammatory bowel disease, including Crohn's disease and ulcerative colitis.
  • [0024]
    From the foregoing, a clear need is apparent for improved compositions and methods to treat malignant conditions and B cell disorders. The compositions and methods of the present invention overcome the limitations of the prior art by providing a binding domain-immunoglobulin fusion protein comprising a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, which is fused to an immunoglobulin heavy chain CH2 constant region polypeptide fused to an immunoglobulin heavy chain CH3 constant region polypeptide, wherein the binding domain-immunoglobulin fusion protein is capable of mediating ADCC or complement fixation. Furthermore, the compositions and methods offer other related advantages.
  • SUMMARY OF THE INVENTION
  • [0025]
    It is an aspect of the present invention to provide a binding domain-immunoglobulin fusion protein, comprising (a) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, wherein said hinge region polypeptide is selected from the group consisting of (i) a mutated hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type immunoglobulin hinge region polypeptide having one or more cysteine residues, (ii) a mutated hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type immunoglobulin hinge region polypeptide having two or more cysteine residues, (iii) a wild-type human IgA hinge region polypeptide, (iv) a mutated human IgA hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type human IgA region polypeptide, and (v) a mutated human IgA hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type human IgA region polypeptide; (b) an immunoglobulin heavy chain CH2 constant region polypeptide that is fused to the hinge region polypeptide; and (c) an immunoglobulin heavy chain CH3 constant region polypeptide that is fused to the CH2 constant region polypeptide, wherein: (1) the binding domain-immunoglobulin fusion protein is capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity and complement fixation, and (2) the binding domain polypeptide is capable of specifically binding to an antigen. In one embodiment the immunoglobulin hinge region polypeptide is a mutated hinge region polypeptide and exhibits a reduced ability to dimerize, relative to a wild-type human immunoglobulin G hinge region polypeptide. In another embodiment the binding domain polypeptide comprises at least one immunoglobulin variable region polypeptide that is an immunoglobulin light chain variable region polypeptide or an immunoglobulin heavy chain variable region polypeptide. In a further embodiment the immunoglobulin variable region polypeptide is derived from a human immunoglobulin.
  • [0026]
    In another embodiment the binding domain Fv-immunoglobulin fusion protein binding domain polypeptide comprises (a) at least one immunoglobulin light chain variable region polypeptide; (b) at least one immunoglobulin heavy chain variable region polypeptide; and (c) at least one linker peptide that is fused to the polypeptide of (a) and to the polypeptide of (b). In a further embodiment the immunoglobulin light chain variable region and heavy chain variable region polypeptides are derived from human immunoglobulins.
  • [0027]
    In another embodiment at least one of the immunoglobulin heavy chain CH2 constant region polypeptide and the immunoglobulin heavy chain CH3 constant region polypeptide is derived from a human immunoglobulin heavy chain. In another embodiment the immunoglobulin heavy chain constant region CH2 and CH3 polypeptides are of an isotype selected from human IgG and human IgA. In another embodiment the antigen is selected from the group consisting of CD19, CD20, CD37, CD40 and L6. In certain further embodiments of the above described fusion protein, the linker polypeptide comprises at least one polypeptide having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21], and in certain other embodiments the linker polypeptide comprises at least three repeats of a polypeptide having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21]. In certain embodiments the immunoglobulin hinge region polypeptide comprises a human IgA hinge region polypeptide. In certain embodiments the binding domain polypeptide comprises a CD154 extracellular domain. In certain embodiments the binding domain polypeptide comprises a CD154 extracellular domain and at least one immunoglobulin variable region polypeptide.
  • [0028]
    In other embodiments the invention provides an isolated polynucleotide encoding any of the above described binding domain-immunoglobulin fusion proteins, and in related embodiments the invention provides a recombinant expression construct comprising such a polynucleotide, and in certain further embodiments the invention provides a host cell transformed or transfected with suc a recombinant expression construct. In another embodiment the invention provides a method of producing a binding domain-immunoglobulin fusion protein, comprising the steps of (a) culturing the host cell as just described, under conditions that permit expression of the binding domain-immunoglobulin fusion protein; and (b) isolating the binding domain-immunoglobulin fusion protein from the host cell culture.
  • [0029]
    The present invention also provides in certain embodiments a pharmaceutical composition comprising a binding domain-immunoglobulin fusion protein as described above, in combination with a physiologically acceptable carrier. In another embodiment there is provided a method of treating a subject having or suspected of having a malignant condition or a B-cell disorder, comprising administering to a patient a therapeutically effective amount of an above described binding domain-immunoglobulin fusion protein. In certain further embodiments the malignant condition or B-cell disorder is a B-cell lymphoma or a disease characterized by autoantibody production, and in certain other further embodiments the malignant condition or B-cell disorder is rheumatoid arthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus, multiple sclerosis or an autoimmune disease.
  • [0030]
    These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • [0031]
    [0031]FIG. 1 shows DNA and deduced amino acid sequences [SEQ ID NOS: ______] of 2H7scFv-Ig, a binding domain-immunoglobulin fusion protein capable of specifically binding CD20.
  • [0032]
    [0032]FIG. 2 shows production levels of 2H7 scFv-Ig by transfected, stable CHO lines and generation of a standard curve by binding of purified 2H7 scFv-Ig to CHO cells expressing CD20.
  • [0033]
    [0033]FIG. 3 shows SDS-PAGE analysis of multiple preparations of isolated 2H7scFv-Ig protein.
  • [0034]
    [0034]FIG. 4 shows complement fixation (FIG. 4A) and mediation of antibody-dependent cellular cytotoxicity (ADCC, FIG. 4B)) by 2H7scFv-Ig.
  • [0035]
    [0035]FIG. 5 shows the effect of simultaneous ligation of CD20 and CD40 on growth of normal B cells.
  • [0036]
    [0036]FIG. 6 shows the effect of simultaneous ligation of CD20 and CD40 on CD95 expression and induction of apoptosis in a B lymphoblastoid cell line.
  • [0037]
    [0037]FIG. 7 shows DNA and deduced amino acid sequences [SEQ ID NOS: ______] of 2H7scFv-CD154 L2 (FIG. 7A, SEQ ID NOS: ______) and 2H7scFv-CD154 S4 (FIG. 7B, SEQ ID NOS: ______ ) binding domain-immunoglobulin fusion proteins capable of specifically binding CD20 and CD40.
  • [0038]
    [0038]FIG. 8 shows binding of 2H7scFv-CD154 binding domain-immunoglobulin fusion proteins to CD20+CHO cells by flow immunocytofluorimetry.
  • [0039]
    [0039]FIG. 9 shows binding of Annexin V to B cell lines Ramos, BJAB, and T51 after binding of 2H7scFv-CD 154 binding domain-immunoglobulin fusion protein to cells.
  • [0040]
    [0040]FIG. 10 shows effects on proliferation of B cell line T51 following binding of 2H7scFv-CD154 binding domain-immunoglobulin fusion protein.
  • [0041]
    [0041]FIG. 11 depicts schematic representations of the structures of 2H7ScFv-Ig fusion proteins [SEQ ID NOS: ______] referred to as CytoxB or CytoxB derivatives: CytoxB-MHWTG1C (2H7 ScFv, mutant hinge, wild-type human IgG1 Fc domain), CytoxB-MHMG1C (2H7 ScFv, mutant hinge, mutated human IgG1 Fc domain) and CytoxB-IgAHWTHG1C (2H7 ScFv, human IgA-derived hinge [SEQ ID NO: ______,] wild-type human IgG1 Fc domain). Arrows indicate position numbers of amino acid residues believed to contribute to FcR binding and ADCC activity (heavy arrows), and to complement fixation (light arrows). Note absence of interchain disulfide bonds.
  • [0042]
    [0042]FIG. 12 shows SDS-PAGE analysis of isolated CytoxB and 2H7scFv-CD 154 binding domain-immunoglobulin fusion proteins.
  • [0043]
    [0043]FIG. 13 shows antibody dependent cell-mediated cytotoxicity (ADCC) activity of CytoxB derivatives.
  • [0044]
    [0044]FIG. 14 shows complement dependent cytotoxicity (CDC) of CytoxB derivatives.
  • [0045]
    [0045]FIG. 15 shows serum half-life determinations of CytoxB-MHWTG1C in macaque blood samples. FIG. 16 shows effects of CytoxB-MHWTG1C on levels of circulating CD40+B cells in macaque blood samples.
  • [0046]
    [0046]FIG. 17 shows production levels of HD37 (CD19-specific) ScFv-Ig by transfected mammalian cell lines and generation of a standard curve by binding of purified HD37 ScFv-Ig to cells expressing CD19.
  • [0047]
    [0047]FIG. 18 shows production levels of L6 (carcinoma antigen) ScFv-Ig by transfected, stable CHO lines and generation of a standard curve by binding of purified L6 ScFv-Ig to cells expressing L6 antigen.
  • [0048]
    [0048]FIG. 19 shows ADCC activity of binding domain-immunoglobulin fusion proteins 2H7 ScFv-Ig, HD37 ScFv-Ig and G28-1 (CD37-specific) ScFv-Ig.
  • [0049]
    [0049]FIG. 20 shows ADCC activity of L6 ScFv-Ig fusion proteins.
  • [0050]
    [0050]FIG. 21 shows SDS-PAGE analysis of L6 ScFv-Ig and 2H7 ScFv-Ig fusion proteins.
  • [0051]
    [0051]FIG. 22 shows SDS-PAGE analysis of G28-1 ScFv-Ig and HD37 ScFv-Ig fusion proteins.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0052]
    The present invention is directed to binding domain-immunoglobulin fusion proteins and to related compositions and methods, which will be useful in immunotherapeutic and immunodiagnostic applications, and which offer certain advantages over antigen-specific polypeptides of the prior art. The fusion proteins of the present invention are preferably single polypeptide chains that comprise, in pertinent part, the following fused domains: a binding domain polypeptide, an immunoglobulin hinge region polypeptide, an immunoglobulin heavy chain CH2 constant region polypeptide, and an immunoglobulin heavy chain CH3 constant region polypeptide. In particularly preferred embodiments, the polypeptide domains of which the binding domain-immunoglobulin fusion protein is comprised are, or are derived from, polypeptides that are the products of human gene sequences, but the invention need not be so limited and may in fact relate to binding domain-immunoglobulin fusion proteins as provided herein that are derived from any natural or artificial source, including genetically engineered and/or mutated polypeptides.
  • [0053]
    The present invention relates in part to the surprising observation that the binding domain-immunoglobulin fusion proteins described herein are capable of immunological activity. More specifically, these proteins retain the ability to participate in well known immunological effector activities including antibody dependent cell mediated cytotoxicity (ADCC, e.g., subsequent to antigen binding on a cell surface, engagement and induction of cytotoxic effector cells bearing appropriate Fc receptors, such as natural killer (NK) cells bearing FcRγIII, under appropriate conditions) and/or complement fixation in complement dependent cytotoxicity (CDC, e.g., subsequent to antigen binding on a cell surface, recruitment and activation of cytolytic proteins that are components of the blood complement cascade ), despite having structures that would not be expected to be capable of promoting such effector activities. As described in greater detail below, ADCC and CDC are unexpected functions for monomeric proteins comprising immunoglobulin heavy chain regions, which are favored by the structures selected for the subject fusion proteins, and particularly by the selection of hinge region polypeptides that are compromised in their ability to form interchain, homodimeric disulfide bonds.
  • [0054]
    Another advantage afforded by the present invention is a binding domain-immunoglobulin fusion polypeptide that can be produced in substantial quantities that are typically greater than those routinely attained with single-chain antibody constructs of the prior art. In preferred embodiments, the binding domain-immunoglobulin fusion polypeptides of the present invention are recombinantly expressed in mammalian expression systems, which offer the advantage of providing polypeptides that are stable in vivo (e.g., under physiological conditions). According to non-limiting theory, such stability may derive in part from posttranslational modifications, and specifically glycosylation, of the fusion proteins. Production of the present binding domain-immunoglobulin fusion proteins via recombinant mammalian expression has been attained in static cell cultures at a level of greater than 50 mg protein per liter culture supernatant and has been routinely observed in such cultures at 10-50 mg/l, such that preferably at least 10-50 mg/l may be produced under static culture conditions; also contemplated are enhanced production of the fusion proteins using art-accepted scale-up methodologies such as “fed batch” (i.e., non-static) production, where yields of at least 5-500 mg/l, and in some instances at least 0.5-1 gm/l, depending on the particular protein product, are obtained.
  • [0055]
    A binding domain polypeptide according to the present invention may be any polypeptide that possesses the ability to specifically recognize and bind to a cognate biological molecule or complex of more than one molecule or assembly or aggregate, whether stable or transient, of such a molecule, which includes a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof. a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any combination thereof such as, for example, a glycoprotein, a glycopeptide, a glycolipid, a lipoprotein, a proteolipid; or any other biological molecule that may be present in a biological sample. Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture, biological fluid or any other tissue or cell preparation from a subject or a biological source. The subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like. In certain preferred embodiments of the invention, the subject or biological source may be suspected of having or being at risk for having a malignant condition or a B-cell disorder as provided herein, which in certain further preferred embodiments may be an autoimmune disease, and in certain other preferred embodiments of the invention the subject or biological source may be known to be free of a risk or presence of such disease.
  • [0056]
    A binding domain polypeptide may therefore be any naturally occurring or recombinantly produced binding partner for a cognate biological molecule as provided herein that is a target structure of interest, herein referred to as an “antigen” but intended according to the present disclosure to encompass any target biological molecule to which it is desirable to have the subject invention fusion protein specifically bind. Binding domain-immunoglobulin fusion proteins are defined to be “immunospecific” or capable of specifically binding if they bind a desired target molecule such as an antigen as provided herein, with a Ka of greater than or equal to about 104 M−1, preferably of greater than or equal to about 105 M−1, more preferably of greater than or equal to about 106 M−1and still more preferably of greater than or equal to about 107 M−1. Affinities of binding domain-immunoglobulin fusion proteins according to the present invention can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Sci. 51:660 (1949). Such determination of fusion protein binding to target antigens of interest can also be performed using any of a number of known methods for identifying and obtaining proteins that specifically interact with other proteins or polypeptides, for example, a yeast two-hybrid screening system such as that described in U.S. Pat. No. 5,283,173 and U.S. Pat. No. 5,468,614, or the equivalent.
  • [0057]
    Preferred embodiments of the subject invention binding domain-immunoglobulin fusion protein comprise binding domains that include at least one immunoglobulin variable region polypeptide, such as all or a portion or fragment of a heavy chain or a light chain V-region, provided it is capable of specifically binding an antigen or other desired target structure of interest as described herein. In other preferred embodiments the binding domain comprises a single chain immunoglobulin-derived Fv product, which may include all or a portion of at least one immunoglobulin light chain V-region and all or a portion of at least one immunoglobulin heavy chain V-region, and which further comprises a linker fused to the V-regions; preparation and testing such constructs are described in greater detail herein and are well known in the art. Other binding domain polypeptides may comprise any protein or portion thereof that retains the ability to specifically bind an antigen as provided herein, including non-immunoglobulins. Accordingly the invention contemplates fusion proteins comprising binding domain polypeptides that are derived from polypeptide ligands such as hormones, cytokines, chemokines, and the like; cell surface or soluble receptors for such polypeptide ligands; lectins; intercellular adhesion receptors such as specific leukocyte integrins, selecting. immunoglobulin gene superfamily members, intercellular adhesion molecules (ICAM-1, -2, -3) and the like; histocompatibility antigens; etc.
  • [0058]
    Examples of cell surface receptors that may provide a binding domain polypeptide, and that may also be selected as the target molecule or antigen to which a binding domain-Ig fusion protein of the present invention desirably binds, include the following, or the like: HER1 (e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO3193), HER2 (Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994Canc. Res. 54:16; see also, e.g., GenBank Ace. Nos. X03363, M17730, SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos. U29339, M34309), HER4 (Plowman et al., 1993 Nature 366:473; see also e.g., GenBank Acc. Nos. L07868, T64105), epidermal growth factor receptor (EGFR) (e.g., GenBank Acc. Nos. U48722, SEG_HEGFREXS, KO3193), vascular endothelial cell growth factor(e.g., GenBank No. M32977), vascular endothelial cell growth factor receptor (e.g., GenBank Acc. Nos. AF022375, 1680143, U48801, X62568), insulin-like growth factor-I (e.g., GenBank Acc. Nos. X00173, X56774, X56773, X06043, see also European Patent No. GB 2241703), insulin-like growth factor-II (e.g., GenBank Ace. Nos. X03562, X00910, SEG_CHUMGFIA, SEG_HUMGFI2, M17863, M17862), transferrin receptor (Trowbridge and Omary, 1981Proc. Nat. Acad. USA 78:3039; see also e.g., GenBank Acc. Nos. X01060, M11507), estrogen receptor (e.g., GenBank Ace. Nos. M38651, X03635, X99101, U47678, M12674), progesterone receptor (e.g., GenBank Ace. Nos. X51730, X69068, M15716), follicle stimulating hormone receptor (FSH-R) (e.g., GenBank Ace. Nos. Z34260, M65085), retinoic acid receptor (e.g., GenBank Ace. Nos. L12060, M60909, X77664, X57280, X07282, X06538), MUC-1 (Barnes et al., 1989Proc. Nat. Acad. Sci. USA 86:7159; see also e.g., GenBank Ace. Nos. SEG_CMUSMUCIO, M65132, M64928) NY-ESO-1 (e.g., GenBank Ace. Nos. AJ003149, U87459), NA 17-A (e.g., European Patent No. WO 96/40039), Melan-A/MART-1 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad. Sci. USA 91:9461; see also e.g., GenBank Acc. Nos. M26729, SEG_HUMTYR 0, see also Weber et al., J. Clin. Invest (1998) 102:1258), Gp-100 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. No. S73003, see also European Patent No. EP 668350; Adema et al., 1994 J. Biol. Chem. 269:20126), MAGE (van den Bruggen et al., 1991 Science 254:1643; see also e.g. GenBank Acc. Nos. U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735, M77481), BAGE (e.g., GenBank Acc. No. U19180, see also U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (e.g., GenBank Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, U19142), any of the CTA class of receptors including in particular HOM-MEL-40 antigen encoded by the SSX2 gene (e.g., GenBank Acc. Nos. X86175, U90842, U90841, X86174), carcinoembyonic antigen (CEA, Gold and Freedman, 1985 J. Exp. Med. 121:439; see also e.g., GenBank Acc. Nos. SEG_HUMCEA, M59710, M59255, M29540), and PyLT (e.g., GenBank Acc. Nos. J02289, J02038).
  • [0059]
    Additional cell surface receptors that may be sources of binding domain polypeptides or that may be cognate antigens include the following, or the like: CD2 (e.g., GenBank Acc. Nos. Y00023, SEG_HUMCD2, M16336, M16445, SEG_MUSCD2, M14362), 4-1BB (CDw137, Kwon et al., 1989 Proc. Nat. Acad. Sci. USA 86:1963, 4-1BB ligand (Goodwin et al., 1993 Eur. J. Immunol. 23:2361; Melero et al., 1998 Eur. J. Immunol. 3:116), CD5 (e.g., GenBank Acc. Nos. X78985, X89405), CD10(e.g., GenBank Acc. Nos. M81591, X76732) CD27 (e.g., GenBank Acc. Nos. M63928, L24495, L08096), CD28 (June et al., 1990 Immunol Today 11:211; see also, e.g., GenBank Acc. Nos. J02988, SEG_HUMCD28, M34563), CTLA-4 (e.g., GenBank Acc. Nos. L 15006, X05719, SEG_HUMIGCTL), CD40 (e.g., GenBank Acc. Nos. M83312, SEG_MUSC040A0, Y10507, X67878, X96710, U15637, L07414), interferon-γ (IFN-γ; see, e.g., Farrar et al. 1993 Ann. Rev. Immunol. 11:571 and references cited therein, Gray et al. 1982 Nature 295:503, Rinderknecht et al. 1984 J. Biol. Chem. 259:6790, DeGrado et al. 1982 Nature 300:379), interleukin-4 (IL-4; see, e.g., 53rd Forum in Immunology, 1993 Research in Immunol. 144:553-643; Banchereau et al., 1994 in The Cytokine Handbook, 2nd ed., A. Thomson, ed., Academic Press, NY, p. 99; Keegan et al., 1994 J. Leukocyt. Biol. 55:272, and references cited therein), interleukin-17 (IL-17) (e.g., GenBank Acc. Nos. U32659, U43088) and interleukin-17 receptor (IL-17R) (e.g., GenBank Acc. Nos. U31993, U58917). Notwithstanding the foregoing, the present invention expressly does not encompass any immunoglobulin fusion protein that is disclosed in U.S. Pat. No. 5,807,734, U.S. Pat. No. 5,795,572 or U.S. Pat. No. 5,807,734.
  • [0060]
    Additional cell surface receptors that may be sources of binding domain polypeptides or that may be cognate antigens include the following, or the like: CD59 (e.g., GenBank Acc. Nos. SEG_HUMCD590, M95708, M34671), CD48 (e.g., GenBank Acc. Nos. M59904), CD58/LFA-3 (e.g.. GenBank Acc. No. A25933, Y00636, E12817; see also JP 1997075090-A) , CD72 (e.g., GenBank Acc. Nos. AA311036, S40777, L35772), CD70 (e.g., GenBank Ace. Nos. Y13636, S69339), CD80/B7.1 (Freeman et al., 1989 J. Immunol. 43:2714; Freeman et al., 1991 J. Exp. Med. 174:625; see also e.g., GenBank Acc. Nos. U33208, 1683379), CD86/B7.2 (Freeman et al., 1993 J. Exp. Med. 178:2185, Boriello et al., 1995 J. Immunol. 155:5490; see also, e.g., GenBank Acc. Nos. AF099105, SEG_MMB72G, U39466, U04343, SEG_HSB725, L25606, L25259), CD40 ligand (e.g., GenBank Acc. Nos. SEG_HUMCD40L, X67878, X65453, L07414), IL-17 (e.g., GenBank Acc. Nos. U32659, U43088), CD43 (e.g., GenBank Acc. Nos. X52075, J04536) and VLA-4 (α4β7) (e.g., GenBank Acc. Nos. L12002, X16983, L20788, U97031, L24913, M68892, M95632). The following cell surface receptors are typically associated with B cells: CD19 (e.g., GenBank Acc. Nos. SEG_HUMCD19W0, M84371, SEG_MUSCD19W, M62542), CD20 (e.g., GenBank Acc. Nos. SEG_HUMCD20, M62541), CD22 (e.g., GenBank Acc. Nos. 1680629, Y10210, X59350, U62631, X52782, L16928), CD30 ligand (e.g., GenBank Acc. Nos. L09753, M83554), CD37 (e.g., GenBank Acc. Nos. SEG_MMCD37X, X14046, X53517), CD106 (VCAM-1) (e.g., GenBank Acc. Nos. X53051, X67783, SEG_MMVCAM1C, see also U.S. Pat. No. 5,596,090), CD54 (ICAM-1) (e.g., GenBank Acc. Nos. X84737, S82847, X06990, J03132, SEG_MUSICAM0), interleukin-12 (see, e.g., Reiter et al, 1993 Crit. Rev. Immunol. 13:1, and references cited therein). Accessory cell agents may also include any of the following cell surface receptors typically associated with dendritic cells: CD83 (e.g., GenBank Acc. Nos. AF001036, AL021918), DEC-205 (e.g., GenBank Acc. Nos. AF011333, U19271).
  • [0061]
    An immunoglobulin hinge region polypeptide, as discussed above, includes any hinge peptide or polypeptide that occurs naturally, as an artificial peptide or as the result of genetic engineering and that is situated in an immunoglobulin heavy chain polypeptide between the amino acid residues responsible for forming intrachain immunoglobulin-domain disulfide bonds in CH1 and CH2 regions; hinge region polypeptides for use in the present invention may also include a mutated hinge region polypeptide. Accordingly, an immunoglobulin hinge region polypeptide may be derived from, or may be a portion or fragment of ( e., one or more amino acids in peptide linkage, typically 5-65 amino acids, preferably 10-50, more preferably 15-35, still more preferably 18-32, still more preferably 20-30, still more preferably 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acids) an immunoglobulin polypeptide chain region classically regarded as having hinge function, as described above, but a hinge region polypeptide for use in the instant invention need not be so restricted and may include amino acids situated (according to structural criteria for assigning a particular residue to a particular domain that may vary, as known in the art) in an adjoining immunoglobulin domain such as a CH1 domain or a CH2 domain, or in the case of certain artificially engineered immunoglobulin constructs, an immunoglobulin variable region domain.
  • [0062]
    Wild-type immunoglobulin hinge region polypeptides include any naturally occurring hinge region that is located between the constant region domains, CH1 and CH2, of an immunoglobulin. The wild-type immunoglobulin hinge region polypeptide is preferably a human immunoglobulin hinge region polypeptide, preferably comprising a hinge region from a human IgG immunoglobulin, and more preferably, a hinge region polypeptide from a human IgG1 isotype. As is known to the art, despite the tremendous overall diversity in immunoglobulin amino acid sequences, immunoglobulin primary structure exhibits a high degree of sequence conservation in particular portions of immunoglobulin polypeptide chains, notably with regard to the occurrence of cysteine residues which, by virtue of their sulfyhydryl groups, offer the potential for disulfide bond formation with other available sulfydryl groups. Accordingly, in the context of the present invention wild-type immunoglobulin hinge region polypeptides may be regarded as those that feature one or more highly conserved (e.g., prevalent in a population in a statistically significant manner) cysteine residues, and in certain preferred embodiments a mutated hinge region polypeptide may be selected that contains zero or one cysteine residue and that is derived from such a wild-type hinge region.
  • [0063]
    A mutated immunoglobulin hinge region polypeptide may comprise a hinge region that has its origin in an immunoglobulin of a species, of an immunoglobulin isotype or class, or of an immunoglobulin subclass that is different from that of the CH2 and CH3 domains. For instance, in certain embodiments of the invention, the binding domain-immunoglobulin fusion protein may comprise a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide comprising a wild-type human IgA hinge region polypeptide, or a mutated human IgA hinge region polypeptide that contains zero or only one cysteine residues, as described herein. Such a hinge region polypeptide may be fused to an immunoglobulin heavy chain CH2 region polypeptide from a different Ig isotype or class, for example an IgG subclass, which in certain preferred embodiments will be the IgG1 subclass.
  • [0064]
    For example, and as described in greater detail below, in certain embodiments of the present invention an immunoglobulin hinge region polypeptide is selected which is derived from a wild-type human IgA hinge region that naturally comprises three cysteines, where the selected hinge region polypeptide is truncated relative to the complete hinge region such that only one of the cysteine residues remains (e.g., SEQ ID NOS: 35-36). Similarly, in certain other embodiments of the invention, the binding domain-immunoglobulin fusion protein comprises a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide comprising a mutated hinge region polypeptide in which the number of cysteine residues is reduced by amino acid substitution or deletion. A mutated hinge region polypeptide may thus be derived from a wild-type immunoglobulin hinge region that contains one or more cysteine residues. In certain embodiments, a mutated hinge region polypeptide may contain zero or only one cysteine residue, wherein the mutated hinge region polypeptide is derived from a wild type immunoglobulin hinge region that contains, respectively, one or more or two or more cysteine residues. In the mutated hinge region polypeptide, the cysteine residues of the wild-type immunoglobulin hinge region are preferably substituted with amino acids that are incapable of forming a disulfide bond. In one embodiment of the invention, the mutated hinge region polypeptide is derived from a human IgG wild-type hinge region polypeptide, which may include any of the four human IgG isotype subclasses, IgG1, IgG2, IgG3 or IgG4. In certain preferred embodiments, the mutated hinge region polypeptide is derived from a human IgG1 wild-type hinge region polypeptide. By way of example, a mutated hinge region polypeptide derived from a human IgG1 wild-type hinge region polypeptide may comprise mutations at two of the three cysteine residues in the wild-type immunoglobulin hinge region, or mutations at all three cysteine residues.
  • [0065]
    The cysteine residues that are present in a wild-type immunoglobulin hinge region and that are removed by mutagenesis according to particularly preferred embodiments of the present invention include cysteine residues that form, or that are capable of forming, interchain disulfide bonds. Without wishing to be bound by theory, the present invention contemplates that mutation of such hinge region cysteine residues, which are believed to be involved in formation of interchain disulfide bridges, reduces the ability of the subject invention binding domain-immunoglobulin fusion protein to dimerize (or form higher oligomers) via interchain disulfide bond formation, while surprisingly not ablating the ability of the fusion protein to promote antibody dependent cell-mediated cytotoxicity (ADCC) or to fix complement. In particular, the Fc receptors (FcR) which mediate ADCC (e.g., FcRIII, CD16) exhibit low affinity for immunoglobulin Fc domains, suggesting that functional binding of Fc to FcR requires avidity stabilization of the Fc-FcR complex by virtue of the dimeric structure of heavy chains in a conventional antibody, and/or FcR aggregation and cross-linking by a conventional Ab Fc structure. (Sonderman et al., 2000 Nature 406:267; Radaev et al., 2001 J. Biol. Chem. 276:16469; Radaev et al., 2001 J. Biol Chem. 276:16478; Koolwijk et al., 1989 J. Immunol, 143:1656; Kato et al., 2000 Immunol. Today 21:310.) Hence, the binding domain-immunoglobulin fusion proteins of the present invention provide the advantages associated with single-chain immunoglobulin fusion proteins while also unexpectedly retaining immunological activity. Similarly, the ability to fix complement is typically associated with immunoglobulins that are dimeric with respect to heavy chain constant regions such as those that comprise Fc, while the binding domain-immunoglobulin fusion proteins of the present invention exhibit the unexpected ability to fix complement.
  • [0066]
    As noted above, binding domain-immunoglobulin fusion proteins are believed, according to non-limiting theory, to be compromised in their ability to dimerize, and further according to theory, this property is a consequence of a reduction in the number of cysteine residues that are present in the immunoglobulin hinge region polypeptide selected for inclusion in the construction of the fusion protein. Determination of the relative ability of a polypeptide to dimerize is well within the knowledge of the relevant art, where any of a number of established methodologies may be applied to detect protein dimerization (see, e.g., Scopes, Protein Purification: Principles and Practice, 1987 Springer-Verlag, New York). For example, biochemical separation techniques for resolving proteins on the basis of molecular size (e.g., gel electrophoresis, gel filtration chromatography, analytical ultracentrifugation, etc.), and/or comparison of protein physicochemical properties before and after introduction of sulfhydryl-active (e.g., iodoacetamide, N-ethylmaleimide) or disulfide-reducing (e.g., 2-mercaptoethanol, dithiothreitol) agents, or other equivalent methodologies, may all be employed for determining a degree of polypeptide dimerization or oligomerization, and for determining possible contribution of disulfide bonds to such potential quarternary structure. In certain embodiments, the invention relates to a binding domain-immunoglobulin fusion protein that exhibits a reduced (i.e., in a statistically significant manner relative to an appropriate IgG-derived control) ability to dimerize, relative to a wild-type human immunoglobulin G hinge region polypeptide as provided herein. Accordingly, those familiar with the art will be able readily to determine whether a particular fusion protein displays such reduced ability to dimerize.
  • [0067]
    Compositions and methods for preparation of immunoglobulin fusion proteins are well known in the art, as described for example, in U.S. Pat. No. 5,892,019, which discloses recombinant antibodies that are the products of a single encoding polynucleotide but which are not binding domain-immunoglobulin fusion proteins according to the present invention.
  • [0068]
    For an immunoglobulin fusion protein of the invention which is intended for use in humans, the constant regions will typically be of human sequence origin, to minimize a potential anti-human immune response and to provide appropriate effector functions. Manipulation of sequences encoding antibody constant regions is described in the PCT publication of Morrison and Oi, WO 89/07142. In particularly preferred embodiments, the CH1 domain is deleted and the carboxyl end of the binding domain, or where the binding domain comprises two immunoglobulin variable region polypeptides, the second (i.e., more proximal to the C-terminus) variable region is joined to the amino terminus of CH2 through the hinge region. A schematic diagram depicting the structures of two exemplary binding domain-immunoglobulin fusion proteins is shown in FIG. 11, where it should be noted that in particularly preferred embodiments no interchain disulfide bonds are present, and in other embodiments a restricted number of interchain disulfide bonds may be present relative to the number of such bonds that would be present if wild-type hinge region polypeptides were instead present, and that in other embodiments the fusion protein comprises a mutated hinge region polypeptide that exhibits a reduced ability to dimerize, relative to a wild-type human IgG hinge region polypeptide. Thus, the isolated polynucleotide molecule codes for a single chain immunoglobulin fusion protein having a binding domain that provides specific binding affinity for a selected antigen.
  • [0069]
    As noted above, in certain embodiments the binding protein-immunoglobulin fusion protein comprises at least one immunoglobulin variable region polypeptide, which may be a light chain or a heavy chain variable region polypeptide, and in certain embodiments the fusion protein comprises at least one such light chain V-region and one such heavy chain V-region and at least one linker peptide that is fused to each of the V-regions. Construction of such binding domains, for example single chain Fv domains, is well known in the art and is described in greater detail in the Examples below, and has been described, for example, in U.S. Pat. Nos. 5,892,019 and references cited therein; selection and assembly of single-chain variable regions and of linker polypeptides that may be fused to each of a heavy chain-derived and a light chain-derived V region (e.g., to generate a binding domain that comprises a single-chain Fv polypeptide) is also known to the art and described herein and, for example, in U.S. Pat. No. 5,869,620, U.S. Pat. No. 4,704,692 and U.S. Pat. No. 4,946,778. In certain embodiments all or a portion of an immunoglobulin sequence that is derived from a non-human source may be “humanized” according to recognized procedures for generating humanized antibodies, i.e., immunoglobulin sequences into which human Ig sequences are introduced to reduce the degree to which a human immune system would perceive such proteins as foreign (see, e.g., U.S. Pat. Nos. 5,693,762; 5,585,089; 4,816,567; 5,225,539; 5,530,101; and references cited therein)
  • [0070]
    Once a binding domain-immunoglobulin fusion protein as provided herein has been designed, DNAs encoding the polypeptide may be synthesized via oligonucleotide synthesis as described, for example, in Sinha et al., Nucleic Acids Res., 12, 4539-4557 (1984); assembled via PCR as described, for example in Innis, Ed., PCR Protocols, Academic Press (1990) and also in Better et al. J. Biol. Chem. 267, 16712-16118 (1992); cloned and expressed via standard procedures as described, for example, in Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989) and also in Robinson et al., Hum. Antibod. Hybridomas, 2, 84-93 (1991); and tested for specific antigen binding activity, as described, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988) and Munson et al., Anal. Biochem., 107, 220-239 (1980).
  • [0071]
    The preparation of single polypeptide chain binding molecules of the Fv region, single-chain Fv molecules, is described in U.S. Pat. No. 4,946,778, which is incorporated herein by reference. In the present invention, single-chain Fv-like molecules are synthesized by encoding a first variable region of the heavy or light chain, followed by one or more linkers to the variable region of the corresponding light or heavy chain, respectively. The selection of appropriate linker(s) between the two variable regions is described in U.S. Pat. No. 4,946,778. An exemplary linker described herein is (Gly-Gly-Gly-Gly-Ser)3. The linker is used to convert the naturally aggregated but chemically separate heavy and light chains into the amino terminal antigen binding portion of a single polypeptide chain, wherein this antigen binding portion will fold into a structure similar to the original structure made of two polypeptide chains and thus retain the ability to bind to the antigen of interest. The nucleotide sequences encoding the variable regions of the heavy and light chains, joined by a sequence encoding a linker, are joined to a nucleotide sequence encoding antibody constant regions. The constant regions are those which permit the resulting polypeptide to form interchain disulfide bonds to form a dimer, and which contain desired effector functions, such as the ability to mediate antibody-dependent cellular cytotoxicity (ADCC). For an immunoglobulin-like molecule of the invention which is intended for use in humans, the constant regions will typically be substantially human to minimize a potential anti-human immune response and to provide approbate effector functions. Manipulation of sequences encoding antibody constant regions is described in the PCT publication of Morrison and Oi, WO 89/07142, which is incorporated herein by reference. In preferred embodiments, the CH1 domain is deleted and the carboxyl end of the second variable region is joined to the amino terminus of CH2 through the hinge region. The Cys residue of the hinge which makes a disulfide bond with a corresponding Cys of the light chain, to hold the heavy and light chains of the native antibody molecule, can be deleted or, preferably, is substituted with, e.g., a Pro residue or the like.
  • [0072]
    As described above, the present invention provides recombinant expression constructs capable of directing the expression of binding domain-immunoglobulin fusion proteins as provided herein. The amino acids, which occur in the various amino acid sequences referred to herein, are identified according to their well known three letter or one letter abbreviations The nucleotides, which occur in the various DNA sequences or fragments thereof referred herein, are designated with the standard single letter designations used routinely in the art. A given amino acid sequence may also encompass similar amino acid sequences having only minor changes, for example by way of illustration and not limitation, covalent chemical modifications, insertions, deletions and substitutions, which may further include conservative substitutions. Amino acid sequences that are similar to one another may share substantial regions of sequence homology. In like fashion, nucleotide sequences may encompass substantially similar nucleotide sequences having only minor changes, for example by way of illustration and not limitation, covalent chemical modifications, insertions, deletions and substitutions, which may further include silent mutations owing to degeneracy of the genetic code. Nucleotide sequences that are similar to one another may share substantial regions of sequence homology.
  • [0073]
    The presence of a malignant condition in a subject refers to the presence of dysplastic, cancerous and/or transformed cells in the subject, including, for example neoplastic, tumor, non-contact inhibited or oncogenically transformed cells, or the like. In preferred embodiments contemplated by the present invention, for example, such cancer cells are malignant hematopoietic cells, such as transformed cells of lymphoid lineage and in particular, B-cell lymphomas and the like; cancer cells may in certain preferred embodiments also be epithelial cells such as carcinoma cells. The invention also contemplates B-cell disorders, which may include certain malignant conditions that affect B-cells (e.g., B-cell lymphoma) but which is not intended to be so limited, and which is also intended to encompass autoimmune diseases and in particular, diseases, disorders and conditions that are characterized by autoantibody production.
  • [0074]
    Autoantibodies are antibodies that react with self antigens. Autoantibodies are detected in several autoimmune diseases (i.e., a disease, disorder or condition wherein a host immune system generates an inappropriate anti-“self” immune reaction) where they are involved in disease activity. The current treatments for these autoimmune diseases are immunosuppressive drugs that require continuing administration, lack specificity, and cause significant side effects. New approaches that can eliminate autoantibody production with minimal toxicity will address an unmet medical need for a spectrum of diseases that affect many people. The subject invention binding domain-immunoglobulin fusion protein is designed for improved penetration into lymphoid tissues. Depletion of B lymphocytes interrupts the autoantibody production cycle, and allows the immune system to reset as new B lymphocytes are produced from precursors in the bone marrow.
  • [0075]
    A number of diseases have been identified for which beneficial effects are believed, according to non-limiting theory, to result from B cell depletion therapy; a brief description of several exemplars of these diseases follows.
  • [0076]
    Autoimmune thyroid disease includes Graves' disease and Hashimoto's thyroiditis. In the United States alone, there are about 20 million people who have some form of autoimmune thyroid disease. Autoimmune thyroid disease results from the production of autoantibodies that either stimulate the thyroid to cause hyperthyroidism (Graves' disease) or destroy the thyroid to cause hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid is caused by autoantibodies that bind and activate the thyroid stimulating hormone (TSH) receptor. Destruction of the thyroid is caused by autoantibodies that react with other thyroid antigens.
  • [0077]
    Current therapy for Graves' disease includes surgery, radioactive iodine, or antithyroid drug therapy. Radioactive iodine is widely used, since antithyroid medications have significant side effects and disease recurrence is high. Surgery is reserved for patients with large goiters or where there is a need for very rapid normalization of thyroid function. There are no therapies that target the production of autoantibodies responsible for stimulating the TSH receptor. Current therapy for Hashimoto's thyroiditis is levothyroxine sodium, and therapy is usually lifelong because of the low likelihood of remission. Suppressive therapy has been shown to shrink goiters in Hashimoto's thryoiditis, but no therapies that reduce autoantibody production to target the disease mechanism are known.
  • [0078]
    Rheumatoid arthritis (RA) is a chronic disease characterized by inflamation of the joints, leading to swelling, pain, and loss of function. RA effects an estimated 2.5 million people in the United States. RA is caused by a combination of events including an initial infection or injury, an abnormal immune response, and genetic factors. While autoreactive T cells and B cells are present in RA, the detection of high levels of antibodies that collect in the joints, called rheumatoid factor, is used in the diagnosis of RA. Current therapy for RA includes many medications for managing pain and slowing the progression of the disease. No therapy has been found that can cure the disease. Medications include nonsteroidal antiinflamatory drugs (NSAIDS), and disease modifying antirheumatic drugs (DMARDS). NSAIDS are effective in benign disease, but fail to prevent the progression to joint destruction and debility in severe RA. Both NSAIDS and DMARDS are associated with signficant side effects. Only one new DMARD, Leflunomide, has been approved in over 10 years. Leflunomide blocks production of autoantibodies, reduces inflamation, and slows progression of RA. However, this drug also causes severe side effects including nausea, diarrhea, hair loss, rash, and liver injury.
  • [0079]
    Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by recurrent injuries to blood vessels in multiple organs, including the kidney, skin, and joints. SLE effects over 500,000 people in the United States. In patients with SLE, a faulty interaction between T cells and B cells results in the production of autoantibodies that attack the cell nucleus. These include anti-double stranded DNA and anti-Sm antibodies. Autoantibodies that bind phospholipids are also found in about half of SLE patients, and are responsible for blood vessel damage and low blood counts. Immune complexes accumulate the kidneys, blood vessels, and joints of SLE patients, where they cause inflamation and tissue damage. No treatment for SLE has been found to cure the disease. NSAIDS and DMARDS are used for therapy depending upon the severity of the disease. Plasmapheresis with plasma exchange to remove autoantibodies can cause temporary improvement in SLE patients. There is general agreement that autoantibodies are responsible for SLE, so new therapies that deplete the B cell lineage, allowing the immune system to reset as new B cells are generated from precursors, offer hope for long lasting benefit in SLE patients.
  • [0080]
    Sjogrens syndrome is an autoimmune disease characterized by destruction of the body's moisture producing glands Sjogrens syndrome is one of the most prevelant autoimmune disorders, striking up to 4 million people in the United States About half of people with Sjogren's also have a connective tissue disease, such as rheumatoid arthritis, while the other half have primary Sjogren's with no other concurrent autoimmune disease. Autoantibodies, including anti-nuclear antibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptor are often present in patients with Sjogrens syndrome. Conventional therapy includes corticosteroids.
  • [0081]
    Immune Thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to blood platelets and cause their destruction. Some cases of ITP are caused by drugs, and others are associated with infection, pregnancy, or autoimmune disease such as SLE. About half of all cases are classified as “idiopathic”, meaning the cause is unknown. The treatment of ITP is determined by the severity of the symptoms. In some cases, no therapy is needed. In most cases, immunosuppressive drugs, including corticosteroids or intravenous infusions of immune globulin to deplete T cells. Another treatment that usually results in an increased number of platelets is removal of the spleen, the organ that destroys antibody-coated platelets. More potent immunosuppressive drugs, including cyclosporine, cyclophosphamide, or azathioprine are used for patients with severe cases. Removal of autoantibodies by passage of patients' plasma over a Protein A column is used as a second line treatment in patients with severe disease.
  • [0082]
    Multiple Sclerosis (MS) is an autoimmune disease characterized by inflamation of the central nervous system and destruction of myelin, which insulates nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are primary contributors to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebral spinal fluid of patients with MS, and some theories predict that the B cell response leading to antibody production is important for mediating the disease. No B cell depletion therapies have been studies in patients with MS. There is no cure for MS. Current therapy is corticosteroids, which can reduce the duration and severity of attacks, but do not affect the course of MS over time. New biotechnology interferon (IFN) therapies for MS have recently been approved.
  • [0083]
    Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorder that is characterized by weakness of the voluntary muscle groups. MG effects about 40,000 people in the United States. MG is caused by autoantibodies that bind to acetylcholine receptors expressed at neuromuscular junctions. The autoantibodies reduce or block acetylcholine receptors, preventing the transmission of signals from nerves to muscles. There is no known cure for MG. Common treatments include immunosuppression with corticosteroids, cyclosporine, cyclophosphamide, or azathioprine. Surgical removal of the thymus is often used to blunt the autoimmune response. Plasmapheresis, used to reduce autoantibody levels in the blood, is effective in MG, but is short-lived because the production of autoantibodies continues. Plasmapheresis is usually reserved for severe muscle weakness prior to surgery.
  • [0084]
    Psoriasis effects approximately five million people. Autoimmune inflamation in the skin. Psoriasis associated with arthritis in 30% (psoriatic arthritis). Many treatments, including steroids, UV light retenoids, vitamin D derivatives, cyclosporine, methotrexate.
  • [0085]
    Scleroderma is a chronic autoimmune disease of the connective tissue that is also known as systemic sclerosis. Scleroderma is characterized by an overproduction of collagen, resulting in a thickening of the skin. Approxiamtely 300,000 people in the United States have scleroderma.
  • [0086]
    Inflamatory Bowel Disease including Crohn's disease and Ulcerative colitis, are autoimmune diseases of the digestive system.
  • [0087]
    The present invention further relates to constructs encoding binding domain-immunoglobulin fusion proteins, and in particular to methods for administering recombinant constructs encoding such proteins that may be expressed, for example, as fragments, analogs and derivatives of such polypeptides. The terms “fragment,” “derivative” and “analog” when referring to binding domain-immunoglobulin fusion polypeptides or fusion proteins, refers to any binding domain-immunoglobulin fusion polypeptide or fusion protein that retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active binding domain-immunoglobulin fusion polypeptide.
  • [0088]
    A fragment, derivative or analog of an binding domain-immunoglobulin fusion polypeptide or fusion protein, including binding domain-immunoglobulin fusion polypeptides or fusion proteins encoded by the cDNAs referred to herein, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which additional amino acids are fused to the binding domain-immunoglobulin fusion polypeptide, including amino acids that are employed for detection or specific functional alteration of the binding domain-immunoglobulin fusion polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • [0089]
    The polypeptides of the present invention include binding domain-immunoglobulin fusion polypeptides and fusion proteins having binding domain polypeptide amino acid sequences that are identical or similar to sequences known in the art, or fragments or portions thereof. For example by way of illustration and not limitation, the human CD154 molecule extracellular domain is contemplated for use according to the instant invention, as are polypeptides having at least 70% similarity (preferably a 70% identity) and more preferably 90% similarity (more preferably a 90% identity) to the reported polypeptide and still more preferably a 95% similarity (still more preferably a 95% identity) to the reported polypeptides and to portions of such polypeptides, wherein such portions of a binding domain-immunoglobulin fusion polypeptide generally contain at least 30 amino acids and more preferably at least 50 amino acids.
  • [0090]
    As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide. Fragments or portions of the nucleic acids encoding polypeptides of the present invention may be used to synthesize full-length nucleic acids of the present invention. As used herein, “% identity” refers to the percentage of identical amino acids situated at corresponding amino acid residue positions when two or more polypeptide are aligned and their sequences analyzed using a gapped BLAST algorithm (e.g., Altschul et al., 1997 Nucl. Ac. Res. 25:3389) which weights sequence gaps and sequence mismatches according to the default weightings provided by the National Institutes of Health/ NCBI database (Bethesda, Md.; see www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).
  • [0091]
    The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • [0092]
    The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).
  • [0093]
    As described herein, the invention provides binding domain-immunoglobulin fusion proteins encoded by nucleic acids that have the binding domain coding sequence fused in frame to an additional immunoglobulin domain encoding sequence to provide for expression of a binding domain polypeptide sequence fused to an additional functional polypeptide sequence that permits, for example by way of illustration and not limitation, detection, functional alteration, isolation and/or purification of the fusion protein. Such fusion proteins may permit functional alteration of a binding domain by containing additional immunoglobulin-derived polypeptide sequences that influence behavior of the fusion product, for example (and as described above) by reducing the availability of sufhydryl groups for participation in disulfide bond formation, and by conferring the ability to potentiate ADCC and/or CDC.
  • [0094]
    Modification of the polypeptide may be effected by any means known to those of skill in this art. The preferred methods herein rely on modification of DNA encoding the fusion protein and expression of the modified DNA. DNA encoding one of the binding domain-immunoglobulin fusions discussed above may be mutagenized using standard methodologies, including those described below. For example, cysteine residues that may otherwise facilitate multimer formation or promote particular molecular conformations can be deleted from a polypeptide or replaced, e.g. cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting protein aggregates in solutions containing physiologically acceptable buffers and salts. In addition, fragments of binding domain-immunoglobulin fusions may be constructed and used. As noted above, the counterreceptor/ ligand binding domains for many candidate binding domain-immunoglobulin fusion have been delineated, such that one having ordinary skill in the art may readily select appropriate polypeptide domains for inclusion in the encoded products of the instant expression constructs.
  • [0095]
    Conservative substitutions of amino acids are well-known and may be made generally without altering the biological activity of the resulting binding domain-immunoglobulin fusion protein molecule. For example, such substitutions are generally made by interchanging within the groups of polar residues, charged residues, hydrophobic residues, small residues, and the like. If necessary, such substitutions may be determined empirically merely by testing the resulting modified protein for the ability to bind to the appropriate cell surface receptors in in vitro biological assays, or to bind to appropriate antigens or desired target molecules.
  • [0096]
    The present invention further relates to nucleic acids which hybridize to binding domain-immunoglobulin fusion protein encoding polynucleotide sequences as provided herein, or their complements, as will be readily apparent to those familiar with the art, if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to nucleic acids which hybridize under stringent conditions to the binding domain-immunoglobulin fusion encoding nucleic acids referred to herein. As used herein, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The nucleic acids which hybridize to binding domain-immunoglobulin fusion encoding nucleic acids referred to herein, in preferred embodiments, encode polypeptides which retain substantially the same biological function or activity as the binding domain-immunoglobulin fusion polypeptides encoded by the cDNAs of the references cited herein.
  • [0097]
    As used herein, to “hybridize” under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules. Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed. Typically “high”, “medium” and “low” stringency encompass the following conditions or equivalent conditions thereto: high stringency: 0.1×SSPE or SSC, 0.1% SDS, 65° C.; medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C.; and low stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C. As known to those having ordinary skill in the art, variations in stringency of hybridization conditions may be achieved by altering the time, temperature and/or concentration of the solutions used for prehybridization, hybridization and wash steps, and suitable conditions may also depend in part on the particular nucleotide sequences of the probe used, and of the blotted, proband nucleic acid sample. Accordingly, it will be appreciated that suitably stringent conditions can be readily selected without undue experimentation where a desired selectivity of the probe is identified, based on its ability to hybridize to one or more certain proband sequences while not hybridizing to certain other proband sequences.
  • [0098]
    The nucleic acids of the present invention, also referred to herein as polynucleotides, may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. A coding sequence which encodes an binding domain-immunoglobulin fusion polypeptide for use according to the invention may be identical to the coding sequence known in the art for any given binding domain-immunoglobulin fusion, or may be a different coding sequence, which, as a result of the redundancy or degeneracy of the genetic code, encodes the same binding domain-immunoglobulin fusion polypeptide.
  • [0099]
    The nucleic acids which encode binding domain-immunoglobulin fusion polypeptides for use according to the invention may include, but are not limited to: only the coding sequence for the binding domain-immunoglobulin fusion polypeptide; the coding sequence for the binding domain-immunoglobulin fusion polypeptide and additional coding sequence; the coding sequence for the binding domain-immunoglobulin fusion polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequences 5′ and/or 3′ of the coding sequence for the binding domain-immunoglobulin fusion polypeptide, which for example may further include but need not be limited to one or more regulatory nucleic acid sequences that may be a regulated or regulatable promoter, enhancer, other transcription regulatory sequence, repressor binding sequence, translation regulatory sequence or any other regulatory nucleic acid sequence. Thus, the term “nucleic acid encoding” or “polynucleotide encoding” a binding domain-immunoglobulin fusion protein encompasses a nucleic acid which includes only coding sequence for a binding domain-immunoglobulin fusion polypeptide as well as a nucleic acid which includes additional coding and/or non-coding sequence(s).
  • [0100]
    Nucleic acids and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g., WO 93/01286, U.S. Application Ser. No. 07/723,454; U.S. Pat. No. 5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124). Identification of oligonucleotides and nucleic acid sequences for use in the present invention involves methods well known in the art. For example, the desirable properties, lengths and other characteristics of useful oligonucleotides are well known. In certain embodiments, synthetic oligonucleotides and nucleic acid sequences may be designed that resist degradation by endogenous host cell nucleolytic enzymes by containing such linkages as: phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages that have proven useful in antisense applications (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec et al., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. Acids Res. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100 (1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246 (1988)).
  • [0101]
    In one embodiment, the present invention provides truncated components (e.g., binding domain polypeptide, hinge region polypeptide, linker, etc.) for use in a binding domain-immunoglobulin fusion protein, and in another embodiment the invention provides nucleic acids encoding a binding domain-immunoglobulin fusion protein having such truncated components. A truncated molecule may be any molecule that comprises less than a full length version of the molecule. Truncated molecules provided by the present invention may include truncated biological polymers, and in preferred embodiments of the invention such truncated molecules may be truncated nucleic acid molecules or truncated polypeptides. Truncated nucleic acid molecules have less than the full length nucleotide sequence of a known or described nucleic acid molecule, where such a known or described nucleic acid molecule may be a naturally occurring, a synthetic or a recombinant nucleic acid molecule, so long as one skilled in the art would regard it as a full length molecule. Thus, for example, truncated nucleic acid molecules that correspond to a gene sequence contain less than the full length gene where the gene comprises coding and non-coding sequences, promoters, enhancers and other regulatory sequences, flanking sequences and the like, and other functional and non-functional sequences that are recognized as part of the gene. In another example, truncated nucleic acid molecules that correspond to a mRNA sequence contain less than the full length mRNA transcript, which may include various translated and non-translated regions as well as other functional and non-functional sequences.
  • [0102]
    In other preferred embodiments, truncated molecules are polypeptides that comprise less than the full length amino acid sequence of a particular protein or polypeptide component. As used herein “deletion” has its common meaning as understood by those familiar with the art, and may refer to molecules that lack one or more of a portion of a sequence from either terminus or from a non-terminal region, relative to a corresponding full length molecule, for example, as in the case of truncated molecules provided herein. Truncated molecules that are linear biological polymers such as nucleic acid molecules or polypeptides may have one or more of a deletion from either terminus of the molecule or a deletion from a non-terminal region of the molecule, where such deletions may be deletions of 1-1500 contiguous nucleotide or amino acid residues, preferably 1-500 contiguous nucleotide or amino acid residues and more preferably 1-300 contiguous nucleotide or amino acid residues. In certain particularly preferred embodiments truncated nucleic acid molecules may have a deletion of 270-330 contiguous nucleotides. In certain other particularly preferred embodiments truncated polypeptide molecules may have a deletion of 80-140 contiguous amino acids.
  • [0103]
    The present invention further relates to variants of the herein referenced nucleic acids which encode fragments, analogs and/or derivatives of a binding domain-immunoglobulin fusion polypeptide. The variants of the nucleic acids encoding binding domain-immunoglobulin fusion may be naturally occurring allelic variants of the nucleic acids or non-naturally occurring variants. As is known in the art, an allelic variant is an alternate form of a nucleic acid sequence which may have at least one of a substitution, a deletion or an addition of one or more nucleotides, any of which does not substantially alter the function of the encoded binding domain-immunoglobulin fusion polypeptide.
  • [0104]
    Variants and derivatives of binding domain-immunoglobulin fusion may be obtained by mutations of nucleotide sequences encoding binding domain-immunoglobulin fusion polypeptides. Alterations of the native amino acid sequence may be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • [0105]
    Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making such alterations are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 12-19, 1985); Smith et al. (Genetic Engineering: Principles and Methods BioTechniques, Jan. 12-19, 1985); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.
  • [0106]
    As an example, modification of DNA may be performed by site-directed mutagenesis of DNA encoding the protein combined with the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension (SOE). Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as M13 phage vectors, which are well-known and commercially available. Other suitable vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (e.g., all or a component portion of a given binding domain-immunoglobulin fusion protein). An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence. The heteroduplex is introduced into appropriate bacterial cells and clones that include the desired mutation are selected. The resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
  • [0107]
    Equivalent DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity are also encompassed by the invention. For example, and as discussed above, sequences encoding Cys residues that are not desirable or essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation.
  • [0108]
    Host organisms include those organisms in which recombinant production of binding domain-immunoglobulin fusion products encoded by the recombinant constructs of the present invention may occur, such as bacteria (for example, E. coli), yeast (for example, Saccharomyces cerevisiae and Pichia pastoris), insect cells and mammals, including in vitro and in vivo expression. Host organisms thus may include organisms for the construction, propagation, expression or other steps in the production of the compositions provided herein; hosts also include subjects in which immune responses take place, as described above. Presently preferred host organisms are E. coli bacterial strains, inbred murine strains and murine cell lines, and human cells, subjects and cell lines.
  • [0109]
    The DNA construct encoding the desired binding domain-immunoglobulin fusion is introduced into a plasmid for expression in an appropriate host. In preferred embodiments, the host is a bacterial host. The sequence encoding the ligand or nucleic acid binding domain is preferably codon-optimized for expression in the particular host. Thus, for example, if a human binding domain-immunoglobulin fusion is expressed in bacteria, the codons would be optimized for bacterial usage. For small coding regions, the gene can be synthesized as a single oligonucleotide. For larger proteins, splicing of multiple oligonucleotides, mutagenesis, or other techniques known to those in the art may be used. The sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription. The sequence of nucleotides encoding a binding domain-immunoglobulin fusion protein may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium.
  • [0110]
    In preferred embodiments, the DNA plasmids also include a transcription terminator sequence. As used herein, a “transcription terminator region” is a sequence that signals transcription termination. The entire transcription terminator may be obtained from a protein-encoding gene, which may be the same or different from the inserted binding domain-immunoglobulin fusion encoding gene or the source of the promoter. Transcription terminators are optional components of the expression systems herein, but are employed in preferred embodiments.
  • [0111]
    The plasmids used herein include a promoter in operative association with the DNA encoding the protein or polypeptide of interest and are designed for expression of proteins in a suitable host as described above (e.g., bacterial, murine or human) depending upon the desired use of the plasmid (e.g., administration of a vaccine containing binding domain-immunoglobulin fusion encoding sequences). Suitable promoters for expression of proteins and polypeptides herein are widely available and are well known in the art. Inducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the trp, lpp, and lac promoters, such as the lacUV5, from E. coli; the P10 or polyhedrin gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and inducible promoters from other eukaryotic expression systems. For expression of the proteins such promoters are inserted in a plasmid in operative linkage with a control region such as the lac operon.
  • [0112]
    Preferred promoter regions are those that are inducible and functional in E. coli. Examples of suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl β-D-thiogalactopyranoside (IPTG; see Nakamura et al., Cell 18:1109-1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g., zinc) induction (see, e.g., U.S. Pat. No. 4,870,009 to Evans et al.); the phage T71ac promoter responsive to IPTG (see, e.g., U.S. Pat. No. 4,952,496; and Studier et al., Meth. Enzymol. 185:60-89, 1990) and the TAC promoter.
  • [0113]
    The plasmids may optionally include a selectable marker gene or genes that are functional in the host. A selectable marker gene includes any gene that confers a phenotype on bacteria that allows transformed bacterial cells to be identified and selectively grown from among a vast majority of untransformed cells. Suitable selectable marker genes for bacterial hosts, for example, include the ampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr) and the kanamycin resistance gene (Kanr). The kanamycin resistance gene is presently preferred.
  • [0114]
    The plasmids may also include DNA encoding a signal for secretion of the operably linked protein. Secretion signals suitable for use are widely available and are well known in the art. Prokaryotic and eukaryotic secretion signals functional in E. coli may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J. Mol. Biol 184:99-105, 1985). In addition, the bacterial pe1B gene secretion signal (Lei et al., J. Bacteriol. 169:4379, 1987), the phoA secretion signal, and the cek2 functional in insect cell may be employed. The most preferred secretion signal is the E. coli ompA secretion signal. Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, e.g., von Heijne, J. Mol. Biol. 184:99-105, 1985). Using the methods described herein, one of skill in the art can substitute secretion signals that are functional in either yeast, insect or mammalian cells to secrete proteins from those cells.
  • [0115]
    Preferred plasmids for transformation of E. coli cells include the pET expression vectors (e.g., pET-11a, pET-12a-c, pET-15b; see U.S. Pat. No. 4,952,496; available from Novagen, Madison, Wis.). Other preferred plasmids include the pKK plasmids, particularly pKK 223-3, which contains the tac promoter (Brosius et al., Proc. Natl. Acad. Sci. 81:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology; U.S. Pat. Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKK has been modified by replacement of the ampicillin resistance gene with a kanamycin resistance gene. (Available from Pharmacia; obtained from pUC4K, see, e.g., Vieira et al. (Gene 19:259-268, 1982; and U.S. Pat. No. 4,719,179.) Baculovirus vectors, such as pBlueBac (also called pJVETL and derivatives thereof), particularly pBlueBac III (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may also be used for expression of the polypeptides in insect cells. Other plasmids include the pIN-IIIompA plasmids (see U.S. Pat. No. 4,575,013; see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such as pIN-IIIompA2.
  • [0116]
    Preferably, the DNA molecule is replicated in bacterial cells, preferably in E. coli. The preferred DNA molecule also includes a bacterial origin of replication, to ensure the maintenance of the DNA molecule from generation to generation of the bacteria. In this way, large quantities of the DNA molecule can be produced by replication in bacteria. Preferred bacterial origins of replication include, but are not limited to, the f1-ori and col E1 origins of replication. Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Pat. No. 4,952,496). Such hosts include, but are not limited to, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLys strains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNA polymerase.
  • [0117]
    The DNA molecules provided may also contain a gene coding for a repressor protein. The repressor protein is capable of repressing the transcription of a promoter that contains sequences of nucleotides to which the repressor protein binds. The promoter can be derepressed by altering the physiological conditions of the cell. For example, the alteration can be accomplished by adding to the growth medium a molecule that inhibits the ability to interact with the operator or with regulatory proteins or other regions of the DNA or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lacI repressor responsive to IPTG induction, the temperature sensitive λ cI857 repressor, and the like. The E coli lacI repressor is preferred.
  • [0118]
    In general, recombinant constructs of the subject invention will also contain elements necessary for transcription and translation. In particular, such elements are preferred where the recombinant expression construct containing nucleic acid sequences encoding binding domain-immunoglobulin fusion proteins is intended for expression in a host cell or organism. In certain embodiments of the present invention, cell type preferred or cell type specific expression of a cell binding domain-immunoglobulin fusion encoding gene may be achieved by placing the gene under regulation of a promoter. The choice of the promoter will depend upon the cell type to be transformed and the degree or type of control desired. Promoters can be constitutive or active and may further be cell type specific, tissue specific, individual cell specific, event specific, temporally specific or inducible. Cell-type specific promoters and event type specific promoters are preferred. Examples of constitutive or nonspecific promoters include the SV40 early promoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No. 5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062), and adenovirus promoter. In addition to viral promoters, cellular promoters are also amenable within the context of this invention. In particular, cellular promoters for the so-called housekeeping genes are useful. Viral promoters are preferred, because generally they are stronger promoters than cellular promoters. Promoter regions have been identified in the genes of many eukaryotes including higher eukaryotes, such that suitable promoters for use in a particular host can be readily selected by those skilled in the art.
  • [0119]
    Inducible promoters may also be used. These promoters include MMTV LTR (PCT WO 91/13160), inducible by dexamethasone; metallothionein promoter, inducible by heavy metals; and promoters with cAMP response elements, inducible by cAMP. By using an inducible promoter, the nucleic acid sequence encoding a binding domain-immunoglobulin fusion protein may be delivered to a cell by the subject invention expression construct and will remain quiescent until the addition of the inducer. This allows further control on the timing of production of the gene product.
  • [0120]
    Event-type specific promoters are active or up-regulated only upon the occurrence of an event, such as tumorigenicity or viral infection. The HIV LTR is a well known example of an event-specific promoter. The promoter is inactive unless the tat gene product is present, which occurs upon viral infection. Some event-type promoters are also tissue-specific.
  • [0121]
    Additionally, promoters that are coordinately regulated with a particular cellular gene may be used. For example, promoters of genes that are coordinately expressed may be used when expression of a particular binding domain-immunoglobulin fusion protein-encoding gene is desired in concert with expression of one or more additional endogenous or exogenously introduced genes. This type of promoter is especially useful when one knows the pattern of gene expression relevant to induction of an immune response in a particular tissue of the immune system, so that specific immunocompetent cells within that tissue may be activated or otherwise recruited to participate in the immune response.
  • [0122]
    In addition to the promoter, repressor sequences, negative regulators, or tissue-specific silencers may be inserted to reduce non-specific expression of binding domain-immunoglobulin fusion protein encoding genes in certain situations, such as, for example, a host that is transiently immunocompromised as part of a therapeutic strategy. Multiple repressor elements may be inserted in the promoter region. Repression of transcription is independent on the orientation of repressor elements or distance from the promoter. One type of repressor sequence is an insulator sequence. Such sequences inhibit transcription (Dunaway et al., Mol Cell Biol 17: 182-9, 1997; Gdula et al., Proc Natl Acad Sci USA 93:9378-83, 1996, Chan et al., J Virol 70: 5312-28, 1996; Scott and Geyer, EMBO J 14:6258-67. 1995; Kalos and Fournier, Mol Cell Biol 15:198-207, 1995; Chung et al., Cell 74: 505-14, 1993) and will silence background transcription.
  • [0123]
    Repressor elements have also been identified in the promoter regions of the genes for type II (cartilage) collagen, choline acetyltransferase, albumin (Hu et al., J. Cell Growth Differ. 3(9):577-588, 1992), phosphoglycerate kinase (PGK-2) (Misuno et al., Gene 119(2):293-297, 1992), and in the 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase gene. (Lemaigre et al., Mol. Cell Biol. 11(2):1099-1106.) Furthermore, the negative regulatory element Tse-1 has been identified in a number of liver specific genes, and has been shown to block cAMP response element—(CRE) mediated induction of gene activation in hepatocytes. (Boshart et al., Cell 61(5):905-916, 1990).
  • [0124]
    In preferred embodiments, elements that increase the expression of the desired product are incorporated into the construct. Such elements include internal ribosome binding sites (IRES; Wang and Siddiqui, Curr. Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top. Microbiol. Immunol. 203:65, 1995; Rees et al., Biotechniques 20:102, 1996; Sugimoto et al., Biotechnology 12:694, 1994). IRES increase translation efficiency. As well, other sequences may enhance expression. For some genes, sequences especially at the 5′ end inhibit transcription and/or translation. These sequences are usually palindromes that can form hairpin structures. Any such sequences in the nucleic acid to be delivered are generally deleted. Expression levels of the transcript or translated product are assayed to confirm or ascertain which sequences affect expression. Transcript levels may be assayed by any known method, including Northern blot hybridization, RNase probe protection and the like. Protein levels may be assayed by any known method, including ELISA, western blot, immunocytochemistry or other well known techniques.
  • [0125]
    Other elements may be incorporated into the binding domain-immunoglobulin fusion protein encoding constructs of the present invention. In preferred embodiments, the construct includes a transcription terminator sequence, including a polyadenylation sequence, splice donor and acceptor sites, and an enhancer. Other elements useful for expression and maintenance of the construct in mammalian cells or other eukaryotic cells may also be incorporated (e.g., origin of replication). Because the constructs are conveniently produced in bacterial cells, elements that are necessary for, or that enhance, propagation in bacteria are incorporated. Such elements include an origin of replication, a selectable marker and the like.
  • [0126]
    As provided herein, an additional level of controlling the expression of nucleic acids encoding binding domain-immunoglobulin fusion proteins delivered to cells using the constructs of the invention may be provided by simultaneously delivering two or more differentially regulated nucleic acid constructs. The use of such a multiple nucleic acid construct approach may permit coordinated regulation of an immune response such as, for example, spatiotemporal coordination that depends on the cell type and/or presence of another expressed encoded component. Those familiar with the art will appreciate that multiple levels of regulated gene expression may be achieved in a similar manner by selection of suitable regulatory sequences, including but not limited to promoters, enhancers and other well known gene regulatory elements.
  • [0127]
    The present invention also relates to vectors, and to constructs prepared from known vectors that include nucleic acids of the present invention, and in particular to “recombinant expression constructs” that include any nucleic acids encoding binding domain-immunoglobulin fusion proteins and polypeptides according to the invention as provided above; to host cells which are genetically engineered with vectors and/or constructs of the invention and to methods of administering expression constructs comprising nucleic acid sequences encoding such binding domain-immunoglobulin fusion polypeptides and fusion proteins of the invention, or fragments or variants thereof, by recombinant techniques. Binding domain-immunoglobulin fusion proteins can be expressed in virtually any host cell under the control of appropriate promoters, depending on the nature of the construct (e.g., type of promoter, as described above), and on the nature of the desired host cell (e.g., whether postmitotic terminally differentiated or actively dividing; e.g., whether the expression construct occurs in host cell as an episome or is integrated into host cell genome). Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989); as noted above, in particularly preferred embodiments of the invention, recombinant expression is conducted in mammalian cells that have been transfected or transformed with the subject invention recombinant expression construct.
  • [0128]
    Typically, the constructs are derived from plasmid vectors. A preferred construct is a modified pNASS vector (Clontech, Palo Alto, Calif.), which has nucleic acid sequences encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see also, e.g., catalogues from Invitrogen, San Diego, Calif.; Novagen, Madison, Wis.; Pharmacia, Piscataway, N.J.; and others). Presently preferred constructs may be prepared that include a dihydrofolate reductase (DHFR) encoding sequence under suitable regulatory control, for promoting enhanced production levels of the binding domain-immunoglobulin fusion protei, which levels result from gene amplification following application of an appropriate selection agent (e.g., methetrexate).
  • [0129]
    Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, as described above. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. Thus, for example, the binding domain-immunoglobulin fusion protein encoding nucleic acids as provided herein may be included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing a binding domain-immunoglobulin fusion polypeptide in a host cell. In certain preferred embodiments the constructs are included in formulations that are administered in vivo. Such vectors and constructs include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies, or replication deficient retroviruses as described below. However, any other vector may be used for preparation of a recombinant expression construct, and in preferred embodiments such a vector will be replicable and viable in the host.
  • [0130]
    The appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); Glover (Ed.) (1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins (Eds.), (1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK); and elsewhere.
  • [0131]
    The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a constitutive promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or their viruses, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding an binding domain-immunoglobulin fusion polypeptide is described herein.
  • [0132]
    Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • [0133]
    As provided herein, in certain embodiments the vector may be a viral vector such as a retroviral vector. (Miller et al., 1989 BioTechniques 7:980; Coffin and Varmus, 1996 Retroviruses, Cold Spring Harbor Laboratory Press, N.Y.) For example, retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • [0134]
    Retroviruses are RNA viruses which can replicate and integrate into the genome of a host cell via a DNA intermediate. This DNA intermediate, or provirus, may be stably integrated into the host cell DNA. According to certain embodiments of the present invention, an expression construct may comprise a retrovirus into which a foreign gene that encodes a foreign protein is incorporated in place of normal retroviral RNA. When retroviral RNA enters a host cell coincident with infection, the foreign gene is also introduced into the cell, and may then be integrated into host cell DNA as if it were part of the retroviral genome. Expression of this foreign gene within the host results in expression of the foreign protein.
  • [0135]
    Most retroviral vector systems which have been developed for gene therapy are based on murine retroviruses. Such retroviruses exist in two forms, as free viral particles referred to as virions, or as proviruses integrated into host cell DNA. The virion form of the virus contains the structural and enzymatic proteins of the retrovirus (including the enzyme reverse transcriptase), two RNA copies of the viral genome, and portions of the source cell plasma membrane containing viral envelope glycoprotein. The retroviral genome is organized into four main regions: the Long Terminal Repeat (LTR),. which contains cis-acting elements necessary for the initiation and termination of transcription and is situated both 5′ and 3′ of the coding genes, and the three coding genes gag, pol, and env. These three genes gag, pol, and env encode, respectively, internal viral structures, enzymatic proteins (such as integrase), and the envelope glycoprotein (designated gp70 and p15e) which confers infectivity and host range specificity of the virus, as well as the “R” peptide of undetermined function.
  • [0136]
    Separate packaging cell lines and vector producing cell lines have been developed because of safety concerns regarding the uses of retroviruses, including their use in expression constructs as provided by the present invention. Briefly, this methodology employs the use of two components, a retroviral vector and a packaging cell line (PCL). The retroviral vector contains long terminal repeats (LTRs), the foreign DNA to be transferred and a packaging sequence (y). This retroviral vector will not reproduce by itself because the genes which encode structural and envelope proteins are not included within the vector genome. The PCL contains genes encoding the gag, pol, and env proteins, but does not contain the packaging signal “y”. Thus, a PCL can only form empty virion particles by itself. Within this general method, the retroviral vector is introduced into the PCL, thereby creating a vector-producing cell line (VCL). This VCL manufactures virion particles containing only the retroviral vector's (foreign) genome, and therefore has previously been considered to be a safe retrovirus vector for therapeutic use.
  • [0137]
    “Retroviral vector construct” refers to an assembly which is, within preferred embodiments of the invention, capable of directing the expression of a sequence(s) or gene(s) of interest, such as binding domain-immunoglobulin fusion encoding nucleic acid sequences. Briefly, the retroviral vector construct must include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis and a 3′ LTR. A wide variety of heterologous sequences may be included within the vector construct, including for example, sequences which encode a protein (e.g., cytotoxic protein, disease-associated antigen, immune accessory molecule, or replacement gene), or which are useful as a molecule itself (e.g., as a ribozyme or antisense sequence).
  • [0138]
    Retroviral vector constructs of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; Rockville, Md.), or isolated from known sources using commonly available techniques. Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral vector constructs, packaging cells, or producer cells of the present invention given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 82:488, 1985).
  • [0139]
    Suitable promoters for use in viral vectors generally may include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g. cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters or promoters as described above.
  • [0140]
    As described above, the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990). The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • [0141]
    The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the binding domain-immunoglobulin fusion polypeptides or fusion proteins. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo, The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the binding domain-immunoglobulin fusion polypeptide or fusion protein. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, circulating peripheral blood mononuclear and polymorphonuclear cells including myelomonocytic cells, lymphocytes, myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoid and reticuloendothelia cells of the lymph nodes and spleen, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • [0142]
    As another example of an embodiment of the invention in which a viral vector is used to prepare the recombinant binding domain-immunoglobulin fusion expression construct, in one preferred embodiment, host cells transduced by a recombinant viral construct directing the expression of binding domain-immunoglobulin fusion polypeptides or fusion proteins may produce viral particles containing expressed binding domain-immunoglobulin fusion polypeptides or fusion proteins that are derived from portions of a host cell membrane incorporated by the viral particles during viral budding.
  • [0143]
    In another aspect, the present invention relates to host cells containing the above described recombinant binding domain-immunoglobulin fusion expression constructs. Host cells are genetically engineered (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention which may be, for example, a cloning vector, a shuttle vector or an expression construct. The vector or construct may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding binding domain-immunoglobulin fusion polypeptides or binding domain-immunoglobulin fusion fusion proteins. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan.
  • [0144]
    The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as E. coli, Streptomycin, Salmonella tvphimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • [0145]
    Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences, for example as described herein regarding the preparation of binding domain-immunoglobulin fusion expression constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al., 1986 Basic Methods in Molecular Biology).
  • [0146]
    The present invention binding domain-immunoglobulin fusion proteins may be formulated into pharmaceutical compositions for administration according to well known methodologies. Pharmaceutical compositions generally comprise one or more recombinant expression constructs, and/or expression products of such constructs, in combination with a pharmaceutically acceptable carrier, excipient or diluent. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. For nucleic acid-based formulations, or for formulations comprising expression products of the subject invention recombinant constructs, about 0.01 μg/kg to about 100 mg/kg body weight will be adminstered, typically by the intradermal, subcutaneous, intramuscular or intravenous route, or by other routes. A preferred dosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kg to about 200 μg/kg particularly preferred. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host. “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.
  • [0147]
    “Pharmaceutically acceptable salt” refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.
  • [0148]
    The pharmaceutical compositions that contain one or more binding domain-immunoglobulin fusion protein encoding constructs (or their expressed products) may be in any form which allows for the composition to be administered to a patient For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e.g., sublingually or buccally), sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intrameatal, intraurethral injection or infusion techniques. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.
  • [0149]
    For oral administration, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose. Coloring and/or flavoring agents may be present. A coating shell may be employed.
  • [0150]
    The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to one or more binding domain-immunoglobulin fusion construct or expressed product, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • [0151]
    A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
  • [0152]
    It may also be desirable to include other components in the preparation, such as delivery vehicles including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of immunostimulatory substances (adjuvants) for use in such vehicles include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon and IL-15.
  • [0153]
    While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, it is preferable that the microsphere be larger than approximately 25 microns.
  • [0154]
    Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.
  • [0155]
    As described above, the subject invention includes compositions capable of delivering nucleic acid molecules encoding binding domain-immunoglobulin fusion proteins. Such compositions include recombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (see Berkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (see U.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)), recombinant expression construct nucleic acid molecules complexed to a polycationic molecule (see WO 93/03709), and nucleic acids associated with liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851, 1987). In certain embodiments, the DNA may be linked to killed or inactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Other suitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989).
  • [0156]
    In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from a host, modified, and placed into the same or another host animal. It will be evident that one can utilize any of the compositions noted above for introduction of binding domain-immunoglobulin fusion proteins or of binding domain-immunoglobulin fusion protein encoding nucleic acid molecules into tissue cells in an ex vivo context. Protocols for viral, physical and chemical methods of uptake are well known in the art.
  • [0157]
    Accordingly, the present invention is useful for treating a patient having a B-cell disorder or a malignant condition, or for treating a cell culture derived from such a patient. As used herein, the term “patient” refers to any warm-blooded animal, preferably a human. A patient may be afflicted with cancer, such as B-cell lymphoma, or may be normal (i.e., free of detectable disease and infection). A “cell culture” is any preparation amenable to ex vivo treatment, for example a preparation containing immunocompetent cells or isolated cells of the immune system (including, but not limited to, T cells, macrophages, monocytes, B cells and dendritic cells). Such cells may be isolated by any of a variety of techniques well known to those of ordinary skill in the art (e.g., Ficoll-hypaque density centrifugation). The cells may (but need not) have been isolated from a patient afflicted with a B-cell disorder or a malignancy, and may be reintroduced into a patient after treatment.
  • [0158]
    A liquid composition intended for either parenteral or oral administration should contain an amount of binding domain-immunoglobulin fusion protein encoding construct or expressed product such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 wt % of a binding domain-immunoglobulin fusion construct or expressed product in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of binding domain-immunoglobulin fusion construct or expressed product(s). Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active compound.
  • [0159]
    The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the binding domain-immunoglobulin fusion construct or expressed product of from about 0.1 to about 10% w/v (weight per unit volume).
  • [0160]
    The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • [0161]
    In the methods of the invention, the binding domain-immunoglobulin fusion encoding constructs or expressed product(s) may be administered through use of insert(s), bead(s), timed-release formulation(s), patch(es) or fast-release formulation(s).
  • [0162]
    The following Examples are offered by way of illustration and not by way of limitation.
  • EXAMPLES Example 1 Cloning of the 2H7 Variable Regions and Construction and Sequencing of 2H7sCFV-IG
  • [0163]
    This Example illustrates the cloning of cDNA molecules that encode the heavy chain and light chain variable regions of the monoclonal antibody 2H7. This Example also demonstrates the construction, sequencing, and expression of 2H7scFv-Ig.
  • [0164]
    Hybridoma cells expressing 2H7 monoclonal antibody that specifically bound to CD20 were provided by Ed Clark at the University of Washington, Seattle, Wash. Prior to harvesting, hybridoma cells were kept in log phase growth for several days in RPMI 1640 media (Life Technologies, Gaithersburg, Md.) supplemented with glutamine, pyruvate, DMEM non-essential amino acids, and penicillin-streptomycin. Cells were pelleted by centrifugation from the culture medium, and 2×107 cells were used to prepare RNA. RNA was isolated from the 2H7-producing hybridoma cells using the Pharmingen (San Diego, Calif.) total RNA isolation kit (Catalog # 45520K) according to the manufacturer's instructions accompanying the kit. One microgram (1 μg) of total RNA was used as template to prepare cDNA by reverse transcription. The RNA and 300 ng random primers were combined and denatured at 72° C. for 10 minutes prior to addition of enzyme. Superscript II reverse transcriptase (Life Technologies) was added to the RNA plus primer mixture in a total volume of 25 μl in the presence of 5× second strand buffer and 0.1 M DTT provided with the enzyme. The reverse transcription reaction was allowed to proceed at 42° C. for one hour.
  • [0165]
    The 2H7 cDNA generated in the randomly primed reverse transcriptase reaction and V region specific primers were used to amplify by PCR the variable regions for the light and heavy chain of the 2H7 antibody. The V region specific primers were designed using the published sequence (Genbank accession numbers M17954 for VL and M17953 for VH) as a guide. The two variable chains were designed with compatible end sequences so that an scFv could be assembled by ligation of the two V regions after amplification and restriction enzyme digestion.
  • [0166]
    A (gly4ser)3 peptide linker to be inserted between the two V regions was incorporated by adding the extra nucleotides to the antisense primer for the VL of 2H7. A Sac I restriction site was also introduced at the junction between the two V regions. The sense primer used to amplify the 2H7 VL, that included a HindIII restriction site and the light chain leader peptide was 5′-gtc aag ctt gcc gcc atg gat ttt caa gtg cag att ttt cag c-3′ (SEQ ID NO:______. The antisense primer was 5′-gtc gtc gag ctc cca cct cet cea gat cca cca ccg ccc gag cca ccg cca cct ttc agc tcc agc ttg gtg cc-3′ (SEQ ID NO:______). The reading frame of the V region is indicated as a bold, underlined codon. The Hind III and SacI sites are indicated by underlined italicized sequences.
  • [0167]
    The VH domain was amplified without a leader peptide, but included a 5′ SacI restriction site for fusion to the VL and a BclI restriction site at the 3′ end for fusion to various tails, including the human IgG1 Fc domain and the truncated forms of CD40 ligand, CD154. The sense primer was 5′-gct gt gag ctc tca ggc tta tct aca gca agt ctg g-3′ (SEQ ID NO:______). The SacI site is indicated in italicized and underlined font, and the reading frame of the codon for the first amino acid of the VH domain is indicated in bold, underlined type. The antisense primer was 5′-gtt gtc tga tca gag acg gtg acc gtg gtc cc-3′ (SEQ ID NO:______). The BclI site is indicated in italicized, underlined type, and the last serine of the VH domain sequence is indicated in bold, underlined type.
  • [0168]
    The scFv-Ig was assembled by inserting the 2H7 scFv HindIII-BclI fragment into pUC 19 containing the human IgG1 hinge, CH2, and CH3 regions, which was digested with restriction enzymes, HindIII and Bell. After ligation, the ligation products were transformed into DHSα bacteria. Positive clones were screened for the properly inserted fragments using the SacI site at the VL-VH junction of 2H7 as a diagnostic site. The 2H7seFv-Ig eDNA was subjected to cycle sequencing on a PE 9700 thermocycler using a 25-cycle program by denaturing at 96° C. for 10 seconds, annealing at 50° C. for 30 seconds, and extending at 72° C. for 4 minutes. The sequencing primers were pUC forward and reverse primers and an internal primer that annealed to the CH2 domain human in the IgG constant region portion. Sequencing reactions were performed using the Big Dye Terminator Ready Sequencing Mix (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Samples were subsequently purified using Centrisep columns (Catalog # CS-901, Princeton Separations, Adelphia, N.J.), the eluates dried in a Savant vacuum dryer, denatured in Template Suppression Reagent (PE-ABI), and analyzed on an ABI 310 Genetic Analyzer (PE-Applied Biosystems). The sequence was edited, translated, and analyzed using Vector Nti version 6.0 (Informax, North Bethesda, Md.). FIG. 1 shows the cDNA and predicted amino acid sequence of the 2H7scFv-Ig construct.
  • Example 2 Expression of 2H7 ScFv-IG in Stable CHO Cell Lines
  • [0169]
    This Example illustrates expression of 2H7scFv-Ig in a eukaryotic cell line and characterization of the expressed 2H7scFv-Ig by SDS-PAGE and by functional assays, including ADCC and complement fixation.
  • [0170]
    The 2H7scFv-Ig HindIII-XbaI (˜1.6 kb) fragment with correct sequence was inserted into the mammalian expression vector pD18, and DNA from positive clones was amplified using QIAGEN plasmid preparation kits (QIAGEN, Valencia, Calif.). The recombinant plasmid DNA (100 μg) was then linearized in a nonessential region by digestion with AscI, purified by phenol extraction, and resuspended in tissue culture media, Excell 302 (Catalog # 14312-79P, JRH Biosciences, Lenexa, Kans.). Cells for transfection, CHO DG44 cells, were kept in logarithmic growth, and 107 cells harvested for each transfection reaction. Linearized DNA was added to the CHO cells in a total volume of 0.8 ml for electroporation.
  • [0171]
    Stable production of the 2H7 scFv-Ig fusion protein (SEQ. ID NO: 10) was achieved by electroporation of a selectable, amplifiable plasmid, pD18, containing the 2H7 scFv-Ig cDNA under the control of the CMV promoter, into Chinese Hamster Ovary (CHO) cells (all cell lines from American Type Culture Collection, Manassas, Va., unless otherwise noted). The 2H7 expression cassette was subcloned downstream of the CMV promoter into the vector multiple cloning site as a ˜1.6 kb HindIII-XbaI fragment. The pD 18 vector is a modified version of pcDNA3 encoding the DHFR selectable marker with an attenuated promoter to increase selection pressure for the plasmid. Plasmid DNA was prepared using Qiagen maxiprep kits, and purified plasmid was linearized at a unique AscI site prior to phenol extraction and ethanol precipitation. Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrier DNA, and 100 μg each of plasmid and carrier DNA was used to transfect 107 CHO DG44 cells by electroporation. Cells were grown to logarithmic phase in Excell 302 media (JRH Biosciences) containing glutamine (4mM), pyruvate, recombinant insulin, penicillin-streptomycin, and 2×DMEM nonessential amino acids (all from Life Technologies, Gaithersburg, Md.), hereafter referred to as “Excell 302 complete” media. Media for untransfected cells also contained HT (diluted from a 100×solution of hypoxanthine and thymidine) (Life Technologies). Media for transfections under selection contained varying levels of methotrexate (Sigma-Aldrich) as selective agent, ranging from 50 nM to 5 μM. Electroporations were performed at 275 volts, 950 μF. Transfected cells were allowed to recover overnight in non-selective media prior to selective plating in 96 well flat bottom plates (Costar) at varying serial dilutions ranging from 125 cells/well to 2000 cells/well Culture media for cell cloning was Excell 302 complete, containing 100 nM methotrexate. Once clonal outgrowth was sufficient, serial dilutions of culture supernatants from master wells were screened for binding to CD20-CHO transfected cells. The clones with the highest production of the fusion protein were expanded into T25 and then T75 flasks to provide adequate numbers of cells for freezing and for scaling up production of the 2H7scFvIg. Production levels were further increased in cultures from three clones by progressive amplification in methotrexate containing culture media. At each successive passage of cells, the Excell 302 complete media contained an increased concentration of methotrexate, such that only the cells that amplified the DHFR plasmid could survive.
  • [0172]
    Supernatants were collected from CHO cells expressing the 2H7scFv-Ig, filtered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.) and were passed over a Protein A-agarose (IPA 300 crosslinked agarose) column (Repligen, Needham, Mass.). The column was washed with PBS, and then bound protein was eluted using 0.1 M citrate buffer, pH 3.0. Fractions were collected and eluted protein was neutralized using 1M Tris, pH 8.0, prior to dialysis overnight in PBS. Concentration of the purified 2H7scFv-Ig (SEQ ID NO:______) was determined by absorption at 280 nm. An extinction coefficient of 1.77 was determined using the protein analysis tools in the Vector Nti Version 6.0 Software package (Informax, North Bethesda, Md.). This program uses the amino acid composition data to calculate extinction coefficients.
  • [0173]
    Production levels of 2H7scFv-Ig by transfected, stable CHO cells were analyzed by flow cytometry. Purified 2H7scFv-Ig to CHO cells was allowed to bind to CHO cells that expressed CD20 (CD20 CHO) and analyzed by flow cytometry using a fluorescein-conjugated anti-human IgG second step reagent (Catalog Numbers H10101 and H10501, CalTag, Burlingame, Calif.). FIG. 2 (top) shows a standard curve generated by titration of 2H7scFv-Ig binding to CD20 CHO. At each concentration of 2H7scFv-Ig, the mean brightness of the fluorescein signal in linear units is shown. Supernatants collected from T flasks containing stable CHO cell clones expressing 2H7scFv-Ig were then allowed to bind to CD20 CHO and the binding was analyzed by flow cytometry. The fluorescein signal generated by 2H7scFv-Ig contained in the supernatants was measured and the 2H7scFv-Ig concentration in the supernatants was calculated from the standard curve (FIG. 2, bottom).
  • [0174]
    Purified 2H7scFv-Ig (SEQ ID NO:______) was analyzed by electrophoresis on SDS-Polyacrylamide gels. Samples of 2H7scFv-Ig, purified by independent Protein A Agarose column runs, were boiled in SDS sample buffer without reduction of disulfide bonds and applied to SDS 10% Tris-BIS gels (Catalog # NP0301, Novex, Carlsbad, Calif.). Twenty micrograms of each purified batch was loaded on the gels. The proteins were visualized after electrophoresis by Coomassie Blue staining (Pierce Gel Code Blue Stain Reagent, Catalog #24590, Pierce, Rockford, Ill.), and destaining in distilled water. Molecular weight markers were included on the same gel (Kaleidoscope Prestained Standards, Catalog # 161-0324, Bio-Rad, Hercules, Calif.). The results are presented in FIG. 3. The numbers above the lanes designate independent purification batches. The molecular weights in kilodaltons of the size markers are indicated on the left side of the figure. Further experiments with alternative sample preparation conditions indicated that reduction of disulfide bonds by boiling the protein in SDS sample buffer containing DTT or 2-mercaptoethanol caused the 2H7scFv-Ig to aggregate.
  • [0175]
    Any number of other immunological parameters may be monitored using routine assays that are well known in the art. These may include, for example, antibody dependent cell-mediated cytotoxicity (ADCC) assays, secondary in vitro antibody responses, flow immunocytofluorimetric analysis of various peripheral blood or lymphoid mononuclear cell subpopulations using well established marker antigen systems, immunohistochemistry or other relevant assays. These and other assays may be found, for example, in Rose et al. (Eds.), Manual of Clinical Laboratory Immunology, 5th Ed., 1997 American Society of Microbiology, Washington, DC.
  • [0176]
    The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence of complement was tested using B cell lines Ramos and Bjab. Rabbit complement (Pel-Freez, Rogers, Ak.) was used in the assay at a final dilution of {fraction (1/10)}. Purified 2H7scFv-Ig was incubated with B cells and complement for 45 minutes at 37° C., followed by counting of live and dead cells by trypan blue exclusion. The results in FIG. 4A show that in the presence of rabbit complement, 2H7scFv-Ig lysed B cells expressing CD20.
  • [0177]
    The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence of peripheral blood mononuclear cells (PBMC) was tested by measuring the release of 51Cr from labeled Bjab cells in a 4-hour assay using a 100:1 ratio of PBMC to Bjab cells. The results shown in FIG. 4B indicated that 2H7scFv-Ig can mediate antibody dependent cellular cytotoxicity (ADCC) because the release of 51Cr was higher in the presence of both PBMC and 2H7scFv-Ig than in the presence of either PBMC or 2H7scFv-Ig alone.
  • Example 3 Effect of Simultaneous Ligation of CD20 and CD40 on Growth of Normal B Cells, and on CD95 Expression, and Induction of Apoptosis
  • [0178]
    This example illustrates the effect of cross-linking of CD20 and CD40 expressed on the cell surface on cell proliferation.
  • [0179]
    Dense resting B cells were isolated from human tonsil by a Percoll step gradient and T cells were removed by E-rosetting. Proliferation of resting, dense tonsillar B cells was measured by uptake of 3[H]-thymidine during the last 12 hours of a 4-day experiment. Proliferation was measured in quadruplicate cultures with means and standard deviations as shown. Murine anti-human CD20 mAb 1F5 (anti-CD20) was used alone or was cross-linked with anti-murine κ mAb 187.1 (anti-CD20XL). CD40 activation was accomplished using soluble human CD 154 fused with murine CD8 (CD 154) (Hollenbaugh et al., EMBO J. 11:4212-21 (1992)), and CD40 cross-linking was accomplished using anti-murine CD8 mAb 53-6 (CD154XL). This procedure allowed simultaneous cross-linking of CD20 and CD40 on the cell surface. The results are presented in FIG. 5.
  • [0180]
    The effect of CD20 and CD40 cross-linking on Ramos cells, a B lymphoma cell line, was examined. Ramos cells were analyzed for CD95 (Fas) expression and percent apoptosis eighteen hours after treatment (no goat anti-mouse IgG (GAM)) and/or cross-linking (+GAM) using murine mAbs that specifically bind CD20 (1F5) and CD40 (G28-5). Control cells were treated with a non-binding isotype control (64.1) specific for CD3.
  • [0181]
    Treated Ramos cells were harvested, incubated with FITC-anti-CD95, and analyzed by flow cytometry to determine the relative expression level of Fas on the cell surface after CD20 or CD40 cross-linking. Data is plotted as mean fluorescence of cells after treatment with the stimuli indicated (FIG. 6A).
  • [0182]
    Treated Ramos cells from the same experiment were harvested and binding of annexin V was measured to indicate the percentage apoptosis in the treated cultures. Apoptosis was measured by binding of Annexin V 18 hours after cross-linking of CD20 and CD40 using 1F5 and G28-5 followed by cross-linking with GAM. Binding of Annexin V was measured using a FITC-Annexin V kit (Catalog # PN-IM2376, Immunotech, Marseille, France,). Annexin V binding is known to be an early event in progression of cells into apoptosis. Apoptosis, or programmed cell death, is a process characterized by a cascade of catabolic reactions leading to cell death by suicide. In the early phase of apoptosis, before cells change morphology and hydrolyze DNA, the integrity of the cell membrane is maintained but cells lose the asymmetry of their membrane phospholipids, exposing negatively charged phospholipids, such as phosphatidylserine, at the cell surface. Annexin V, a calcium and phopholipid binding protein, binds preferentially and with high affinity to phosphatidylserine. Results demonstrating the effect of cross-linking both CD20 and CD40 on expression of the FAS receptor (CD95) are presented in FIG. 6B. The effect of cross-linking of both CD20 and CD40 on Annexin V binding to cells is shown in FIG. 6B.
  • Example 4 Construction and Characterization of 2H7 ScFv-CD 154 Fusion Proteins
  • [0183]
    To construct a molecule capable of binding to both CD20 and CD40, cDNA encoding the 2H7 scFv was fused with EDNA encoding CD154, the CD40 ligand. The 2H7 scFv cDNA encoded on the HindIII-BclI fragment was removed from the 2H7 scFvIg construct, and inserted into a pD18 vector along with a BamHI-XbaI cDNA fragment encoding the extracellular domain of human CD154. The extracellular domain is encoded at the carboxy terminus of CD154, similar to other type II membrane proteins.
  • [0184]
    The extracellular domain of human CD154 was PCR amplified using CDNA generated with random primers and RNA from human T lymphocytes activated with PHA (phytohemagglutinin). The primer sets included two different 5′ or sense primers that created fusion junctions at two different positions within the extracellular domain of CD154. Two different fusion junctions were designed that resulted in a short or truncated form (form S4) including amino acids 108 (Glu)−261 (Leu)+(Glu), and a long or complete form (form L2) including amino acids 48 (Ark)−261 (Leu)+(Glu), of the extracellular domain of CD154, both constructed as BamHI-XbaI fragments. The sense primer which fuses the two different truncated extracellular domains to the 2H7scFv includes a BamHI site for cloning. The sense primer for the S4 form of the CD154 cDNA is designated SEQUENCE ID NO: 11 or CD154BAM108 and encodes a 34 mer with the following sequence : 5′-gtt gtc gga tcc aga aaa cag ctt tga aat gca a-3′, while the antisense primer is designated SEQUENCE ID NO: 12 or CD154XBA and encodes a 44 mer with the following sequence: 5′-gtt gtt tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3′.
  • [0185]
    The oligonucleotide primers used in amplifying the long form (L2) of the CD154 extracellular domain encoding amino acids 48 (Arg)−261 (Leu)+(Glu), were as follows: The sense primer designated CD154 BAM48 (SEQUENCE ID NO: 13) encoded a 35-mer with the following sequence: 5′-gtt gtc gga tcc aag aag gtt gga caa gat aga ag-3′. The antisense primer designated or CD154XBA (SEQUENCE ID NO: ______) encoded the 44-mer: 5′-gtt gtt tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3′. Other PCR reaction conditions were identical to those used for amplifying the 2H7 scFv (see Example 1) PCR fragments were purified by PCR quick kits (QIAGEN, San Diego, Calif.), eluted in 30 μl ddH2O, and digested with BamHI and XbaI (Roche) restriction endonucleases in a 40 μl reaction volume at 37° C. for 3 hours. Fragments were gel purified, purified using QIAEX kits according to the manufacturer's instructions (QIAGEN), and ligated along with the 2H7 HindIII-BclI fragment into the pD18 expression vector digested with HindIII+XbaI. Ligation reactions were transformed into DH5-alpha chemically competent bacteria and plated onto LB plates containing 100 μg/ml ampicillin. Transformants were grown overnight at 37° C., and isolated colonies used to inoculate 3 ml liquid cultures in Luria Broth containing 100 μg/ml ampicillin. Clones were screened after mini-plasmid preparations (QIAGEN) for insertion of both the 2H7 scFv and the CD154 extracellular domain fragments.
  • [0186]
    The 2H7scFv-CD154 construct cDNAs were subjected to cycle sequencing on a PE 9700 thermocycler using a 25-cycle program that included denaturating at 96° C., 10 seconds, annealing at 50° C. for 5 seconds, and extension at 60° C., for 4 minutes, The sequencing primers used were pD18 forward (SEQ ID NO: ______: 5′-gtctatataagcagagctctggc-3′ ) and pD18 reverse (SEQ ID NO: ______: 5′-cgaggctgatcagcgagctctagca-3′ ) primers. In addition, an internal primer was used that had homology to the human CD154 sequence (SEQ ID NO: ______: 5′-ccgcaatttgaggattctgatcacc-3′ ). Sequencing reactions included primers at 3.2 pmol, approximately 200 ng DNA template, and 8 μl sequencing mix. Sequencing reactions were performed using the Big Dye Terminator Ready Sequencing Mix (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Samples were subsequently purified using Centrisep columns (Princeton Separations, Adelphia, N.J.). The eluates were dried in a Savant speed-vacuum dryer, denatured in 20 μl template Suppression Reagent (ABI) at 95° C. for 2 minutes, and analyzed on an ABI 310 Genetic Analyzer (PE-Applied Biosystems). The sequence was edited, translated, and analyzed using Vector Nti version 6.0 (Informax, North Bethesda, Md.). The 2H7scFv-CD154 L2 cDNA sequence and predicted amino acid sequence is presented in FIG. 7A, and 2H7scFv-CD154 S4 cDNA sequence and predicted amino acid sequence is presented in FIG. 7B.
  • [0187]
    The binding activity of the 2H7 scFv-CD154 fusion proteins (SEQ. ID NO: ______ and _______) to CD20 and CD40 simultaneously was determined by flow cytometry. The assay used CHO cell targets that express CD20. After a 45-minute incubation of CD20 CHO cells with supernatants from cells transfected with the 2H7 scFv-CD154 expression plasmid, the CD20 CHO cells were washed twice and incubated with biotin-conjugated CD40-Ig fusion protein in PBS/2% FBS. After 45 min, cells were washed twice and incubated with phycoerythrin (PE)-labeled strepavidin at 1:100 in PBS/2% FBS (Molecular Probes, Eugene Oreg.). After an additional 30 min incubation, cells were washed 2× and were analyzed by flow cytometry. The results show that the 2H7 scFv-CD154 molecule was able to bind to CD20 on the cell surface and to capture biotin-conjugated CD40 from solution (FIG. 8).
  • [0188]
    To determine the effect of the 2H7scFv-CD154 on growth and viability of B lymphoma and lymphoblastoid cell lines, cells were incubated with 2H7scFv-CD154 L2 (SEQ. ID NO: ______) for 12 hours and then examined for binding of Annexin V. Binding of Annexin V was measured using a FITC-Annexin V kit (Immunotech, Marseille, France, Catalog # PN-IM2376). B cell lines were incubated in 1 ml cultures with dilutions of concentrated, dialyzed supernatants from cells expressing secreted forms of the 2H7scFv-CD 154 fusion proteins. The results are presented in FIG. 9.
  • [0189]
    The growth rate of the Ramos B lymphoma cell line in the presence of 2H7scFv-CD 154 was examined by uptake of 3H-thymidine for the last 6 hours of a 24-hour culture. The effect of 2H7scFv-CD154 on cell proliferation is shown in FIG. 10.
  • Example 5 Construction and Characterization of Cytoxb Antibody Derivatives
  • [0190]
    CytoxB antibodies were derived from the 2H7 scFv-IgG polypeptide. The 2H7 scFv (see Example 1) was linked to the human IgG1 Fc domain via an altered hinge domain (see FIG. 11). Cysteine residues in the hinge region were substituted with serine residues by site-directed mutagenesis and other methods known in the art. The mutant hinge was fused either to a wild-type Fc domain to create one construct, designated CytoB-MHWTG1C, or was fused to a mutated Fc domain (CytoxB-MHMG1C) that had additional mutations introduced into the CH2 domain. Amino acid residues in CH2 that are implicated in effector function are illustrated in FIG. 11. Mutations of one or more of these residues may reduce FcR binding and mediation of effector functions. In this example, the leucine residue 234 known in the art to be important to Fc receptor binding, was mutated in the 2H7 scFv fusion protein, CytoxB-[MG1H/MG1C]. In another construct, the human IgG1 hinge region was substituted with a portion of the human IgA hinge, which was fused to wild-type human Fc domain (CytoxB-IgAHWTHG1C). (See FIG. 11). This mutated hinge region allows expression of a mixture of monomeric and dimeric molecules that retain functional properties of the human IgG1 CH2 and CH3 domains. Synthetic, recombinant cDNA expression cassettes for these molecules were constructed and polypeptides were expressed in CHODG44 cells according to methods described in Example 2.
  • [0191]
    Purified fusion protein derivatives of CytoxB-scFvIg molecules were analyzed by SDS-PAGE according to the methods described in Example 2. Polyacrylamide gels were run under non-reducing and reducing conditions. Two different molecule weight marker sets, BioRad prestained markers, (BioRad, Hercules, Calif.) and Novex Multimark molecular weight markers were loaded onto each gel. The migration patterns of the different constructs and of Rituximab™ are presented in FIG. 12.
  • [0192]
    The ability of the different derivatives of CytoxB-scFvIg molecules to mediated ADCC was measured using the Bjab B lymphoma cells as the target and freshly prepared human PBMCs as effector cells, (See Example 2). Effector to target ratios were varied as follows: 70:1, 35:1, and 18:1, with the number of Bjab cells per well remaining constant but the number of PBMCs were varied. Bjab cells were labeled for 2 hours with 51Cr and aliquoted at a cell density of 5×104 cells/well to each well of flat-bottom 96 well plates. Purified fusion proteins or rituximab were added at a concentration of 10 mg/ml to the various dilutions of PBMCs. Spontaneous release was measured without addition of PBMC or fusion protein, and maximal release was measured by the addition of detergent (1% NP-40) to the appropriate wells. Reactions were incubated for 4 hours, and 100 μl of culture supernatant was harvested to a Lumaplate (Packard Instruments) and allowed to dry overnight prior to counting cpm released. The results are presented in FIG. 13.
  • [0193]
    Complement dependent cytotoxicity (CDC) activity of the CytoxB derivatives was also measured. Reactions were performed essentially as described in Example 2. The results are presented in FIG. 14 as percent of dead cells to total cells for each concentration of fusion protein.
  • Example 6 In Vivo Studies in Macaques
  • [0194]
    Initial in vivo studies with CytoxB derivatives have been performed in nonhuman primates. FIG. 15 shows data characterizing the serum half-life of CytoxB in monkeys. Measurements were performed on serum samples obtained from two different macaques (J99231 and K99334) after doses of 6 mg/kg were administered to each monkey on the days indicated by arrows. For each sample, the level of 2H7scFvIg present was estimated by comparison to a standard curve generated by binding of purified CytoxB-(MHWTG1C)-Ig fusion protein to CD20 CHO cells (see Example 2). The data are tabulated in the bottom panel of the FIG. 15.
  • [0195]
    The effect of CytoxB-(MHWTG1C)Ig fusion protein on levels of circulating CD40+ cells in macaques was investigated. Complete blood counts were performed at each of the days indicated in FIG. 16. In addition, FACS (fluorescence activated cell sorter) assays were performed on peripheral blood lymphocytes using a CD40-specific fluorescein conjugated antibody to detect B cells among the cell population. The percentage of positive cells was then used to calculate the number of B cells in the original samples. The data are graphed as thousands of B cells per microliter of blood measured at the days indicated after injection (FIG. 16).
  • Example 7 Construction and Expression of an Anti-Cd19 scFv-IG Fusion Protein
  • [0196]
    An anti-CD19 scFv-Ig fusion protein was constructed, transfected into eukaryotic cells, and expressed according to methods presented in Examples 1, 2, and 5 and standard in the art. The variable heavy chain regions and variable light chain regions were cloned from RNA isolated from hybridoma cells producing antibody HD37, which specifically binds to CD19. Expression levels of a HD37scFv-IgAHWTG1C and a HD37scFv-IgMHWTG1C were measured and compared to a standard curve generated using purified HD37 scFvIg. The results are presented in FIG. 17.
  • Example 8 Construction and Expression of an Anti-L6 scFv-IG Fusion Protein
  • [0197]
    An scFv-Ig fusion protein was constructed using variable regions derived from an anti-carcinoma mAb, L6. The fusion protein was constructed, transfected into eukaryotic cells, and expressed according to methods presented in Examples 1, 2, and 5 and standard in the art. Expression levels of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C were measured and compared to a standard curve generated using purified HD37 scFvIg. The results are presented in FIG. 18.
  • Example 9 Characterization of Various scFv-IG Fusion Proteins
  • [0198]
    In addition to the scFv-Ig fusion protein already described, G28-1 (anti-CD37) scFv-Ig fusion proteins were prepared essentially as described in Examples 1 and 5. The variable regions of the heavy and light chains were cloned according to methods known in the art. ADCC activity of 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1 IgAHWTG1C, HD37- MHWTG1C, and HD37- IgAHWTG1C was determined according to methods described above (see Example 2). Results are presented in FIG. 19. ADCC activity of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C was measured using the 2981 human lung carcinoma cell line. The results are presented in FIG. 20. The murine L6 monoclonal antibody is known not to exhibit ADCC activity.
  • [0199]
    The purified proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions. Samples were prepared and gels run essentially as described in Examples 2 and 5. The results for the L6 and 2H7 scFv-Ig fusion proteins are presented in FIG. 21 and the results for the G28-1 and HD37 scFv-Ig fusion proteins are presented in FIG. 22.
  • [0200]
    From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 38
    <210> SEQ ID NO 1
    <211> LENGTH: 812
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE SCFV FUSION GENE
    <221> NAME/KEY: sig_peptide
    <222> LOCATION: (13)..(78)
    <221> NAME/KEY: V_region
    <222> LOCATION: (79)..(396)
    <223> OTHER INFORMATION: light chain variable region for anti-CD20 scFv
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (397)..(444)
    <223> OTHER INFORMATION: asp-gly3ser(gly4ser)2-ser peptide linker
    <221> NAME/KEY: V_region
    <222> LOCATION: (445)..(808)
    <223> OTHER INFORMATION: heavy chain variable region for anti-CD20 scFv
    <400> SEQUENCE: 1
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaaggtg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat ca 812
    <210> SEQ ID NO 2
    <211> LENGTH: 1518
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE HUMAN CHIMERIC FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(807)
    <223> OTHER INFORMATION: MURINE ANTI-HUMAN CD20 scFv
    <221> NAME/KEY: C_region
    <222> LOCATION: (808)..(1513)
    <223> OTHER INFORMATION: HUMAN IgG1 Fc TAIL, WILD TYPE HINGE, CH2 AND
    CH3
    <400> SEQUENCE: 2
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat caggagccca aatcttgtga caaaactcac 840
    acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 900
    ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960
    gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020
    cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080
    gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140
    aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200
    gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260
    ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320
    gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380
    ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440
    tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500
    ccgggtaaat gatctaga 1518
    <210> SEQ ID NO 3
    <211> LENGTH: 1518
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE-HUMAN CHIMERIC FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(807)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV
    <221> NAME/KEY: C_region
    <222> LOCATION: (808)..(1513)
    <223> OTHER INFORMATION: HINGE CYSTEINES (826-829; 844-847; 853-856)
    MUTATED TO SERINES
    PROLINE TO SERINE MUTATION (880-883) IN CH2 DISRUPTS EFFECTOR FUN
    CTION
    <400> SEQUENCE: 3
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat caggagccca aatcttctga caaaactcac 840
    acatccccac cgtccccagc acctgaactc ctggggggat cgtcagtctt cctcttcccc 900
    ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960
    gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020
    cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080
    gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140
    aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200
    gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260
    ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320
    gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380
    ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440
    tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500
    ccgggtaaat gatctaga 1518
    <210> SEQ ID NO 4
    <211> LENGTH: 1518
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE-HUMAN CHIMERIC FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(807)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV
    <221> NAME/KEY: C_region
    <222> LOCATION: (808)..(1513)
    <223> OTHER INFORMATION: HINGE CYSTEINES MUTATED TO SERINES (826-829;
    844-847;853-856)
    WILD TYPE CH2 AND CH3 DOMAINS MEDIATE EFFECTOR FUNCTIONS
    <400> SEQUENCE: 4
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat caggagccca aatcttctga caaaactcac 840
    acatccccac cgtccccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 900
    ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960
    gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020
    cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080
    gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140
    aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200
    gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260
    ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320
    gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380
    ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440
    tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500
    ccgggtaaat gatctaga 1518
    <210> SEQ ID NO 5
    <211> LENGTH: 1524
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE HUMAN CHIMERIC FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(796)
    <223> OTHER INFORMATION: MOUSE ANTI HUMAN CD20 SCFV
    <221> NAME/KEY: N_region
    <222> LOCATION: (797)..(864)
    <223> OTHER INFORMATION: HUMAN IGA HINGE REGION
    <221> NAME/KEY: C_region
    <222> LOCATION: (865)..(1518)
    <223> OTHER INFORMATION: HUMAN IGG1 CH2 AND CH3 WILD TYPE FC DOMAIN
    <400> SEQUENCE: 5
    atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataattgcc 60
    agaggacaaa ttgttctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120
    gtcacaatga cttgcagggc cagctcaagt gtaagttaca tgcactggta ccagcagaag 180
    ccaggatcct cccccaaacc ctggatttat gccccatcca acctggcttc tggagtccct 240
    gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagagtggag 300
    gctgaagatg ctgccactta ttactgccag cagtggagtt ttaacccacc cacgttcggt 360
    gctgggacca agctggagct gaaagatggc ggtggctcgg gcggtggtgg atctggagga 420
    ggtgggagct ctcaggctta tctacagcag tctggggctg agctggtgag gcctggggcc 480
    tcagtgaaga tgtcctgcaa ggcttctggc tacacattta ccagttacaa tatgcactgg 540
    gtaaagcaga cacctagaca gggcctggaa tggattggag ctatttatcc aggaaatggt 600
    gatacttcct acaatcagaa gttcaagggc aaggccacac tgactgtaga caaatcctcc 660
    agcacagcct acatgcagct cagcagcctg acatctgaag actctgcggt ctatttctgt 720
    gcaagagtgg tgtactatag taactcttac tggtacttcg atgtctgggg cacagggacc 780
    acggtcaccg tctctgatca gccagttccc tcaactccac ctaccccatc tccctcaact 840
    ccacctaccc catctccctc atgcgcacct gaactcctgg ggggaccgtc agtcttcctc 900
    ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 960
    gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 1020
    gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1080
    gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1140
    gtctccaaca aagccctccc agcccccatc gagaaaacaa tctccaaagc caaagggcag 1200
    ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1260
    gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1320
    agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1380
    tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1440
    ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1500
    ctgtctccgg gtaaatgatc taga 1524
    <210> SEQ ID NO 6
    <211> LENGTH: 711
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC HUMAN PARTIAL FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(705)
    <223> OTHER INFORMATION: HINGE CYSTEINES MUTATED TO SERINES (19-21; 37-
    39; 46-48)
    <400> SEQUENCE: 6
    gatcaggagc ccaaatcttc tgacaaaact cacacatccc caccgtcccc agcacctgaa 60
    ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120
    tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180
    aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240
    gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300
    ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360
    aaaacaatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420
    tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480
    cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540
    acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600
    aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660
    aaccactaca cgcagaagag cctctccctg tctccgggta aatgatctag a 711
    <210> SEQ ID NO 7
    <211> LENGTH: 729
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC HUMAN PARTIAL FUSION GENE
    <221> NAME/KEY: N_region
    <222> LOCATION: (1)..(69)
    <223> OTHER INFORMATION: HUMAN IGA HINGE
    <221> NAME/KEY: C_region
    <222> LOCATION: (70)..(723)
    <223> OTHER INFORMATION: HUMAN WILD TYPE IGG1 CH2 AND CH3, FC
    <400> SEQUENCE: 7
    gatcagccag ttccctcaac tccacctacc ccatctccct caactccacc taccccatct 60
    ccctcatgcg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 120
    aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 180
    cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 240
    aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 300
    gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 360
    ctcccagccc ccatcgagaa aacaatctcc aaagccaaag ggcagccccg agaaccacag 420
    gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 480
    ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 540
    gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 600
    agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 660
    atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 720
    tgatctaga 729
    <210> SEQ ID NO 8
    <211> LENGTH: 825
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE SCFV FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(72)
    <223> OTHER INFORMATION: LIGHT CHAIN LEADER PEPTIDE
    <221> NAME/KEY: V_region
    <222> LOCATION: (73)..(405)
    <223> OTHER INFORMATION: LIGHT CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD19: HD37
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (406)..(450)
    <223> OTHER INFORMATION: SYNTHETIC (GLY4SER)3 LINKER PEPTIDE
    <221> NAME/KEY: V_region
    <222> LOCATION: (454)..(825)
    <223> OTHER INFORMATION: HEAVY CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD19: HD37
    <400> SEQUENCE: 8
    aagcttgccg ccatggagac agacacactc ctgctatggg tgctgctgct ctgggttcca 60
    ggctccactg gtgacattgt gctgacccaa tctccagctt ctttggctgt gtctctaggg 120
    cagagggcca ccatctcctg caaggccagc caaagtgttg attatgatgg tgatagttat 180
    ttgaactggt accaacagat tccaggacag ccacccaaac tcctcatcta tgatgcatcc 240
    aatctagttt ctgggatccc acccaggttt agtggcagtg ggtctgggac agacttcacc 300
    ctcaacatcc atcctgtgga gaaggtggat gctgcaacct atcactgtca gcaaagtact 360
    gaggatccgt ggacgttcgg tggaggcacc aagctggaaa tcaaaggtgg cggtggctcg 420
    ggcggtggtg ggtcgggtgg cggcggatcg tcacaggttc agctgcagca gtctggggct 480
    gagctggtga ggcctgggtc ctcagtgaag atttcctgca aggcttctgg ctatgcattc 540
    agtagctact ggatgaactg ggtgaagcag aggcctggac agggtcttga gtggattgga 600
    cagatttggc ctggagatgg tgatactaac tacaatggaa agttcaaggg taaagccact 660
    ctgactgcag acgaatcctc cagcacagcc tacatgcaac tcagcagcct agcatctgag 720
    gactctgcgg tctatttctg tgcaagacgg gagactacga cggtaggccg ttattactat 780
    gctatggact actggggtca aggaacctca gtcaccgtct cctca 825
    <210> SEQ ID NO 9
    <211> LENGTH: 795
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE SCFV FUSION GENE
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(72)
    <223> OTHER INFORMATION: LIGHT CHAIN LEADER PEPTIDE SEQUENCE
    <221> NAME/KEY: V_region
    <222> LOCATION: (73)..(393)
    <223> OTHER INFORMATION: LIGHT CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD37: G28-1
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (394)..(441)
    <223> OTHER INFORMATION: SYNTHETIC LINKER PEPTIDE ENCODED (GLY4SER)3
    <221> NAME/KEY: V_region
    <222> LOCATION: (442)..(795)
    <223> OTHER INFORMATION: HEAVY CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD37: G28-1
    <400> SEQUENCE: 9
    aagcttgccg ccatggtatc cacagctcag ttccttgggt tgctgctgct gtggcttaca 60
    ggtggcagat gtgacatcca gatgactcag tctccagcct ccctatctgc atctgtggga 120
    gagactgtca ccatcacatg tcgaacaagt gaaaatgttt acagttattt ggcttggtat 180
    cagcagaaac agggaaaatc tcctcagctc ctggtctctt ttgcaaaaac cttagcagaa 240
    ggtgtgccat caaggttcag tggcagtgga tcaggcacac agttttctct gaagatcagc 300
    agcctgcagc ctgaagattc tggaagttat ttctgtcaac atcattccga taatccgtgg 360
    acgttcggtg gaggcaccga actggagatc aaaggtggcg gtggctcggg cggtggtggg 420
    tcgggtggcg gcggatcgtc agcggtccag ctgcagcagt ctggacctga gctggaaaag 480
    cctggcgctt cagtgaagat ttcctgcaag gcttctggtt actcattcac tggctacaat 540
    atgaactggg tgaagcagaa taatggaaag agccttgagt ggattggaaa tattgatcct 600
    tattatggtg gtactaccta caaccggaag ttcaagggca aggccacatt gactgtagac 660
    aaatcctcca gcacagccta catgcagctc aagagtctga catctgagga ctctgcagtc 720
    tattactgtg caagatcggt cggccctatg gactactggg gtcaaggaac ctcagtcacc 780
    gtctcttctg atcag 795
    <210> SEQ ID NO 10
    <211> LENGTH: 824
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC MOUSE FUSION GENE
    <221> NAME/KEY: sig_peptide
    <222> LOCATION: (1)..(61)
    <223> OTHER INFORMATION: native light chain leader peptide
    <221> NAME/KEY: V_region
    <222> LOCATION: (62)..(397)
    <223> OTHER INFORMATION: LIGHT CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD22:G28-7
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (398)..(445)
    <223> OTHER INFORMATION: (gly4ser)3 linker peptide
    <221> NAME/KEY: V_region
    <222> LOCATION: (445)..(818)
    <223> OTHER INFORMATION: HEAVY CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD22:G28-7
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (819)..(824)
    <223> OTHER INFORMATION: BclI restriction site
    <400> SEQUENCE: 10
    atggagtcac attcccaggt ctttctctcc ctgctgctct gggtatctgg tacctgtggg 60
    aacattatga tgacacagtc gccatcatct ctggctgtgt cagcaggaga aaaggtcact 120
    atgaactgta agtccagtca aagtgttttc tacagttcaa atcagaggaa ttatttggcc 180
    tggtatcagc agaaaccagg gcagtctccc aaattgctga tctactgggc atctactagg 240
    gaatctggtg tccctgatcg cttcacaggc agtggatccg ggacagactt tactcttacc 300
    atcagcagtg tacatactga agacctggca gtttattact gtcatcaatt cctctcttcg 360
    tggacgttcg gtggaggcac caagctggaa atcaaaggcg gtggtggttc gggtggtggt 420
    ggttcgggtg gcggcggatc ttctcaggtc caactgcagc agcctggggc tgaactggtg 480
    aagcctggga cttcagtgaa gctgtcctgc aaggcctctg gctacacctt caccaactac 540
    tggatggtct gggtgaagca gacgcctgga gaaggccttg agtggattgg agaaattatt 600
    cctagcaacg gtcgtactaa atacaatgag aagttcaaga gcaaggccac actgactgca 660
    gacaaatcct cccgcacagc ctacatgcaa ctcagcagcc tggcatctga ggactctgcg 720
    gtctattatt gtgcaagaga gatgtccatt attactacgg tactgactcc cggtttgctt 780
    actggggcca agggactctg gtcactgtct ctgcagcctg atca 824
    <210> SEQ ID NO 11
    <211> LENGTH: 266
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: INIT_MET
    <222> LOCATION: (1)..(1)
    <221> NAME/KEY: SIGNAL
    <222> LOCATION: (1)..(22)
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (23)..(128)
    <223> OTHER INFORMATION: LIGHT CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD20
    <221> NAME/KEY: SITE
    <222> LOCATION: (129)..(144)
    <223> OTHER INFORMATION: ASP-(GLY3SER)-(GLY4SER)2-SER LINKER PEPTIDE
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (145)..(266)
    <223> OTHER INFORMATION: HEAVY CHAIN VARIABLE DOMAIN FOR MOUSE ANTI-
    HUMAN CD20
    <400> SEQUENCE: 11
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp
    260 265
    <210> SEQ ID NO 12
    <211> LENGTH: 271
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(271)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD19 SCFV
    <400> SEQUENCE: 12
    Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
    1 5 10 15
    Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
    20 25 30
    Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser
    35 40 45
    Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro
    50 55 60
    Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser
    65 70 75 80
    Gly Ile Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
    85 90 95
    Leu Asn Ile His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys
    100 105 110
    Gln Gln Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
    115 120 125
    Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
    130 135 140
    Gly Ser Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg
    145 150 155 160
    Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe
    165 170 175
    Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
    180 185 190
    Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn
    195 200 205
    Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser
    210 215 220
    Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
    225 230 235 240
    Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr
    245 250 255
    Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
    260 265 270
    <210> SEQ ID NO 13
    <211> LENGTH: 259
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(259)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD37 SCFV
    <400> SEQUENCE: 13
    Met Val Ser Thr Ala Gln Phe Leu Gly Leu Leu Leu Leu Trp Leu Thr
    1 5 10 15
    Gly Gly Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
    20 25 30
    Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn
    35 40 45
    Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
    50 55 60
    Gln Leu Leu Val Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser
    65 70 75 80
    Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser
    85 90 95
    Ser Leu Gln Pro Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser
    100 105 110
    Asp Asn Pro Trp Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys Gly
    115 120 125
    Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ala
    130 135 140
    Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser
    145 150 155 160
    Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Asn
    165 170 175
    Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile Gly
    180 185 190
    Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe Lys
    195 200 205
    Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
    210 215 220
    Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
    225 230 235 240
    Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
    245 250 255
    Val Ser Ser
    <210> SEQ ID NO 14
    <211> LENGTH: 272
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(272)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD22 SCFV
    <400> SEQUENCE: 14
    Met Glu Ser His Ser Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser
    1 5 10 15
    Gly Thr Cys Gly Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala
    20 25 30
    Val Ser Ala Gly Glu Lys Val Thr Met Asn Cys Lys Ser Ser Gln Ser
    35 40 45
    Val Phe Tyr Ser Ser Asn Gln Arg Asn Tyr Leu Ala Trp Tyr Gln Gln
    50 55 60
    Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
    65 70 75 80
    Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
    85 90 95
    Phe Thr Leu Thr Ile Ser Ser Val His Thr Glu Asp Leu Ala Val Tyr
    100 105 110
    Tyr Cys His Gln Phe Leu Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys
    115 120 125
    Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
    130 135 140
    Gly Gly Ser Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
    145 150 155 160
    Lys Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
    165 170 175
    Phe Thr Asn Tyr Trp Met Val Trp Val Lys Gln Thr Pro Gly Glu Gly
    180 185 190
    Leu Glu Trp Ile Gly Glu Ile Ile Pro Ser Asn Gly Arg Thr Lys Tyr
    195 200 205
    Asn Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
    210 215 220
    Arg Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala
    225 230 235 240
    Val Tyr Tyr Cys Ala Arg Glu Met Ser Ile Ile Thr Thr Val Leu Thr
    245 250 255
    Pro Gly Leu Leu Thr Gly Ala Lys Gly Leu Trp Ser Leu Ser Leu Gln
    260 265 270
    <210> SEQ ID NO 15
    <211> LENGTH: 499
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(265)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV: 2H7
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (266)..(499)
    <223> OTHER INFORMATION: HUMAN IGG1 WILD TYPE HINGE, CH2, CH3 FC
    <400> SEQUENCE: 15
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gln Glu Pro Lys Ser Cys
    260 265 270
    Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
    275 280 285
    Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
    290 295 300
    Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
    305 310 315 320
    Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
    325 330 335
    His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
    340 345 350
    Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
    355 360 365
    Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
    370 375 380
    Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
    385 390 395 400
    Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
    405 410 415
    Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
    420 425 430
    Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
    435 440 445
    Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
    450 455 460
    Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
    465 470 475 480
    His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
    485 490 495
    Pro Gly Lys
    <210> SEQ ID NO 16
    <211> LENGTH: 499
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(265)
    <223> OTHER INFORMATION: 2H7 SCFV TARGETED TO HUMAN CD20
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (265)..(499)
    <223> OTHER INFORMATION: HINGE CYSTEINES MUTATED TO SERINES (AMINO ACIDS
    272 278, 281)
    PROLINE IN CH2 MUTATED TO SERINE (AMINO ACID 290)
    <400> SEQUENCE: 16
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gln Glu Pro Lys Ser Ser
    260 265 270
    Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly
    275 280 285
    Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
    290 295 300
    Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
    305 310 315 320
    Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
    325 330 335
    His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
    340 345 350
    Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
    355 360 365
    Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
    370 375 380
    Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
    385 390 395 400
    Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
    405 410 415
    Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
    420 425 430
    Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
    435 440 445
    Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
    450 455 460
    Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
    465 470 475 480
    His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
    485 490 495
    Pro Gly Lys
    <210> SEQ ID NO 17
    <211> LENGTH: 499
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(265)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV: 2H7
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (266)..(499)
    <223> OTHER INFORMATION: HINGE CYSTEINES MUTATED TO SERINES (AMINO ACIDS
    272,278, 281)
    CH2 AND CH3 DOMAINS ARE WILD TYPE IN SEQUENCE
    <400> SEQUENCE: 17
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gln Glu Pro Lys Ser Ser
    260 265 270
    Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly
    275 280 285
    Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
    290 295 300
    Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
    305 310 315 320
    Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
    325 330 335
    His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
    340 345 350
    Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
    355 360 365
    Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
    370 375 380
    Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
    385 390 395 400
    Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
    405 410 415
    Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
    420 425 430
    Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
    435 440 445
    Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
    450 455 460
    Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
    465 470 475 480
    His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
    485 490 495
    Pro Gly Lys
    <210> SEQ ID NO 18
    <211> LENGTH: 505
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(265)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV: 2H7
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (266)..(288)
    <223> OTHER INFORMATION: WILD TYPE IGA HINGE
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (289)..(505)
    <223> OTHER INFORMATION: HUMAN IGG1 CH2 AND CH3 DOMAINS, WILD TYPE
    SEQUENCE
    <400> SEQUENCE: 18
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gln Pro Val Pro Ser Thr
    260 265 270
    Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys
    275 280 285
    Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
    290 295 300
    Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
    305 310 315 320
    Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
    325 330 335
    Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
    340 345 350
    Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
    355 360 365
    Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
    370 375 380
    Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
    385 390 395 400
    Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
    405 410 415
    Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
    420 425 430
    Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
    435 440 445
    Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
    450 455 460
    Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
    465 470 475 480
    Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
    485 490 495
    Lys Ser Leu Ser Leu Ser Pro Gly Lys
    500 505
    <210> SEQ ID NO 19
    <211> LENGTH: 234
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (1)..(234)
    <223> OTHER INFORMATION: MUTANT IGG1 HINGE (AMINO ACIDS 7, 13, 16)
    WILD TYPE CH2 AND CH3 DOMAINS
    ALTERNATIVE CARBOXY TERMINUS OF SCFVIG FUSION PROTEINS
    <400> SEQUENCE: 19
    Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser
    1 5 10 15
    Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
    20 25 30
    Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
    35 40 45
    Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
    50 55 60
    Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
    65 70 75 80
    Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
    85 90 95
    His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
    100 105 110
    Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
    115 120 125
    Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
    130 135 140
    Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
    145 150 155 160
    Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
    165 170 175
    Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
    180 185 190
    Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
    195 200 205
    Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
    210 215 220
    Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
    225 230
    <210> SEQ ID NO 20
    <211> LENGTH: 240
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(23)
    <223> OTHER INFORMATION: ALTERNATIVE CARBOXY TERMINUS OF SCFVIG FUSION .
    PROTEINS
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (24)..(240)
    <223> OTHER INFORMATION: HUMAN IGG1 WILD TYPE CH2 AND CH3 FC
    <400> SEQUENCE: 20
    Asp Gln Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro
    1 5 10 15
    Pro Thr Pro Ser Pro Ser Cys Ala Pro Glu Leu Leu Gly Gly Pro Ser
    20 25 30
    Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
    35 40 45
    Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
    50 55 60
    Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
    65 70 75 80
    Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
    85 90 95
    Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
    100 105 110
    Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
    115 120 125
    Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
    130 135 140
    Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
    145 150 155 160
    Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
    165 170 175
    Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
    180 185 190
    Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
    195 200 205
    Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
    210 215 220
    Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
    225 230 235 240
    <210> SEQ ID NO 21
    <211> LENGTH: 1470
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(808)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (814)..(1455)
    <223> OTHER INFORMATION: HUMAN EXTRACELLULAR DOMAIN LONG FORM, CD154
    <400> SEQUENCE: 21
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat ccaagaaggt tggacaagat agaagatgaa 840
    aggaatcttc atgaagattt tgtattcatg aaaacgatac agagatgcaa cacaggagaa 900
    agatccttat ccttactgaa ctgtgaggag attaaaagcc agtttgaagg ctttgtgaag 960
    gatataatgt taaacaaaga ggagacgaag aaagaaaaca gctttgaaat gcaaaaaggt 1020
    gatcagaatc ctcaaattgc ggcacatgtc ataagtgagg ccagcagtaa aacaacatct 1080
    gtgttacagt gggctgaaaa aggatactac accatgagca acaacttggt aaccctggaa 1140
    aatgggaaac agctgaccgt taaaagacaa ggactctatt atatctatgc ccaagtcacc 1200
    ttctgttcca atcgggaagc ttcgagtcaa gctccattta tagccagcct ctgcctaaag 1260
    tcccccggta gattcgagag aatcttactc agagctgcaa atacccacag ttccgccaaa 1320
    ccttgcgggc aacaatccat tcacttggga ggagtatttg aattgcaacc aggtgcttcg 1380
    gtgtttgtca atgtgactga tccaagccaa gtgagccatg gcactggctt cacgtccttt 1440
    ggcttactca aactcgagtg ataatctaga 1470
    <210> SEQ ID NO 22
    <211> LENGTH: 1290
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (13)..(808)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (814)..(1275)
    <223> OTHER INFORMATION: HUMAN EXTRACELLULAR DOMAIN, SHORT FORM, CD154
    <400> SEQUENCE: 22
    aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60
    gtcataattg ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120
    ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180
    taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240
    tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300
    agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360
    cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420
    ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagctggtg 480
    aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540
    aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600
    ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac actgactgta 660
    gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga agactctgcg 720
    gtctatttct gtgcaagagt ggtgtactat agtaactctt actggtactt cgatgtctgg 780
    ggcacaggga ccacggtcac cgtctctgat ccagaaaaca gctttgaaat gcaaaaaggt 840
    gatcagaatc ctcaaattgc ggcacatgtc ataagtgagg ccagcagtaa aacaacatct 900
    gtgttacagt gggctgaaaa aggatactac accatgagca acaacttggt aaccctggaa 960
    aatgggaaac agctgaccgt taaaagacaa ggactctatt atatctatgc ccaagtcacc 1020
    ttctgttcca atcgggaagc ttcgagtcaa gctccattta tagccagcct ctgcctaaag 1080
    tcccccggta gattcgagag aatcttactc agagctgcaa atacccacag ttccgccaaa 1140
    ccttgcgggc aacaatccat tcacttggga ggagtatttg aattgcaacc aggtgcttcg 1200
    gtgtttgtca atgtgactga tccaagccaa gtgagccatg gcactggctt cacgtccttt 1260
    ggcttactca aactcgagtg ataatctaga 1290
    <210> SEQ ID NO 23
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 23
    gtcaagcttg ccgccatgga ttttcaagtg cagatttttc agc 43
    <210> SEQ ID NO 24
    <211> LENGTH: 74
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 24
    gtcgtcgagc tcccacctcc tccagatcca ccaccgcccg agccaccgcc acctttcagc 60
    tccagcttgg tccc 74
    <210> SEQ ID NO 25
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 25
    gctgctgagc tctcaggctt atctacagca agtctgg 37
    <210> SEQ ID NO 26
    <211> LENGTH: 32
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 26
    gttgtctgat cagagacggt gaccgtggtc cc 32
    <210> SEQ ID NO 27
    <211> LENGTH: 34
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 27
    gttgtcggat ccagaaaaca gctttgaaat gcaa 34
    <210> SEQ ID NO 28
    <211> LENGTH: 44
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 28
    gttgtttcta gattatcact cgagtttgag taagccaaag gacg 44
    <210> SEQ ID NO 29
    <211> LENGTH: 35
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 29
    gttgtcggat ccaagaaggt tggacaagat agaag 35
    <210> SEQ ID NO 30
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 30
    gtctatataa gcagagctct ggc 23
    <210> SEQ ID NO 31
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 31
    cgaggctgat cagcgagctc tagca 25
    <210> SEQ ID NO 32
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: OLIGONUCLEOTIDE
    <400> SEQUENCE: 32
    ccgcaatttg aggattctga tcacc 25
    <210> SEQ ID NO 33
    <211> LENGTH: 482
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(266)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN CD20 SCFV
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (268)..(481)
    <223> OTHER INFORMATION: EXTRACELLULAR DOMAIN, LONG FORM, HUMAN CD154
    <400> SEQUENCE: 33
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Pro Arg Arg Leu Asp Lys
    260 265 270
    Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val Phe Met Lys Thr
    275 280 285
    Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys
    290 295 300
    Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu
    305 310 315 320
    Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly
    325 330 335
    Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser
    340 345 350
    Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met
    355 360 365
    Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys
    370 375 380
    Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn
    385 390 395 400
    Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys
    405 410 415
    Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His
    420 425 430
    Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val
    435 440 445
    Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro
    450 455 460
    Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys
    465 470 475 480
    Leu Glu
    <210> SEQ ID NO 34
    <211> LENGTH: 422
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MOUSE-HUMAN HYBRID FUSION PROTEIN
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)..(266)
    <223> OTHER INFORMATION: MOUSE ANTI-HUMAN SCFV
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (268)..(421)<223> EXTRACELLULAR DOMAIN, SHORT FORM,
    HUMAN CD154
    <400> SEQUENCE: 34
    Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
    1 5 10 15
    Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
    20 25 30
    Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
    35 40 45
    Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser
    50 55 60
    Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
    65 70 75 80
    Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
    85 90 95
    Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
    100 105 110
    Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
    115 120 125
    Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
    130 135 140
    Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
    145 150 155 160
    Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
    165 170 175
    Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile
    180 185 190
    Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
    195 200 205
    Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
    210 215 220
    Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
    225 230 235 240
    Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
    245 250 255
    Gly Thr Gly Thr Thr Val Thr Val Ser Asp Pro Glu Asn Ser Phe Glu
    260 265 270
    Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
    275 280 285
    Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
    290 295 300
    Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
    305 310 315 320
    Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
    325 330 335
    Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
    340 345 350
    Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
    355 360 365
    Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
    370 375 380
    Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
    385 390 395 400
    Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
    405 410 415
    Gly Leu Leu Lys Leu Glu
    420
    <210> SEQ ID NO 35
    <211> LENGTH: 63
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: N_region
    <222> LOCATION: (1)..(63)
    <223> OTHER INFORMATION: PORTION OF HUMAN IGA HINGE DOMAIN CONTAINING
    ONLY 1 CYSTEINE
    <400> SEQUENCE: 35
    ccagttccct caactccacc taccccatct ccctcaactc cacctacccc atctccctca 60
    tgc 63
    <210> SEQ ID NO 36
    <211> LENGTH: 21
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 36
    Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr
    1 5 10 15
    Pro Ser Pro Ser Cys
    20
    <210> SEQ ID NO 37
    <211> LENGTH: 763
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(6)
    <223> OTHER INFORMATION: BCLI SITE FOR FUSION TO AMIN TERMINAL SCFVS
    <221> NAME/KEY: N_region
    <222> LOCATION: (8)..(752)
    <223> OTHER INFORMATION: WILD TYPE IGA HINGE, CH2, CH3 DOMAINS
    TRUNCATED TO REMOVE SECRETORY COMPONENT ATTACHMENT
    <400> SEQUENCE: 37
    tgatcagcca gttccctcaa ctccacctac cccatctccc tcaactccac ctaccccatc 60
    tccctcatgc tgccaccccc gactgtcact gcaccgaccg gccctcgagg acctgctctt 120
    aggttcagaa gcgatcctca cgtgcacact gaccggcctg agagatgcct caggtgtcac 180
    cttcacctgg acgccctcaa gtgggaagag cgctgttcaa ggaccacctg accgtgacct 240
    ctgtggctgc tacagcgtgt ccagtgtcct gccgggctgt gccgagccat ggaaccatgg 300
    gaagaccttc acttgcactg ctgcctaccc cgagtccaag accccgctaa ccgccaccct 360
    ctcaaaatcc ggaaacacat tccggcccga ggtccacctg ctgccgccgc cgtcggagga 420
    gctggccctg aacgagctgg tgacgctgac gtgcctggca cgtggcttca gccccaagga 480
    tgtgctggtt cgctggctgc aggggtcaca ggagctgccc cgcgagaagt acctgacttg 540
    ggcatcccgg caggagccca gccagggcac caccaccttc gctgtgacca gcatactgcg 600
    cgtggcagcc gaggactgga agaaggggga caccttctcc tgcatggtgg gccacgaggc 660
    cctgccgctg gccttcacac agaagaccat cgaccgcttg gcgggtaaac ccacccatgt 720
    caatgtgtct gttgtcatgg cggaggtgga ctgataatct aga 763
    <210> SEQ ID NO 38
    <211> LENGTH: 250
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: DOMAIN
    <222> LOCATION: (3)..(250)
    <223> OTHER INFORMATION: TRUNCATED FORM, REMOVAL OF LAST THREE AMINO
    ACIDS THAT MEDIATE ATTACHMENT TO SECRETORY COMPONEN
    <400> SEQUENCE: 38
    Asp Gln Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro
    1 5 10 15
    Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser Leu His Arg
    20 25 30
    Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Ile Leu Thr Cys
    35 40 45
    Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe Thr Trp Thr
    50 55 60
    Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Asp Arg Asp Leu
    65 70 75 80
    Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu Pro
    85 90 95
    Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser
    100 105 110
    Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg
    115 120 125
    Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn
    130 135 140
    Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp
    145 150 155 160
    Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys
    165 170 175
    Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly Thr Thr Thr
    180 185 190
    Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp Trp Lys Lys
    195 200 205
    Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu Pro Leu Ala
    210 215 220
    Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro Thr His Val
    225 230 235 240
    Asn Val Ser Val Val Met Ala Glu Val Asp
    245 250

Claims (22)

What is claimed is:
1. A binding domain-immunoglobulin fusion protein, comprising:
(a) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, wherein said hinge region polypeptide is selected from the group consisting of (i) a mutated hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type immunoglobulin hinge region polypeptide having one or more cysteine residues, (ii) a mutated hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type immunoglobulin hinge region polypeptide having two or more cysteine residues. (iii) a wild-type human IgA hinge region polypeptide, (iv) a mutated human IgA hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type human IgA region polypeptide, and (v) a mutated human IgA hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type human IgA region polypeptide;
(b) an immunoglobulin heavy chain CH2 constant region polypeptide that is fused to the hinge region polypeptide; and
(c) an immunoglobulin heavy chain CH3 constant region polypeptide that is fused to the CH2 constant region polypeptide, wherein:
(1) the binding domain-immunoglobulin fusion protein is capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity and complement fixation, and
(2) the binding domain polypeptide is capable of specifically binding to an antigen.
2. The binding domain-immunoglobulin fusion protein of claim 1 wherein the immunoglobulin hinge region polypeptide is a mutated hinge region polypeptide and exhibits a reduced ability to dimerize, relative to a wild-type human immunoglobulin G hinge region polypeptide.
3. The binding domain-immunoglobulin fusion protein of claim 1 wherein the binding domain polypeptide comprises at least one immunoglobulin variable region polypeptide that is selected from the group consisting of an immunoglobulin light chain variable region polypeptide and an immunoglobulin heavy chain variable region polypeptide.
4. The binding domain -immunoglobulin fusion protein of claim 3 wherein the immunoglobulin variable region polypeptide is derived from a human immunoglobulin.
5. The binding domain Fv-immunoglobulin fusion protein of claim 1 wherein the binding domain polypeptide comprises:
(a) at least one immunoglobulin light chain variable region polypeptide;
(b) at least one immunoglobulin heavy chain variable region polypeptide; and
(c) at least one linker peptide that is fused to the polypeptide of (a) and to the polypeptide of (b).
6. The binding domain-immunoglobulin fusion protein of claim 5 wherein the immunoglobulin light chain variable region and heavy chain variable region polypeptides are derived from human immunoglobulins.
7. The binding domain-immunoglobulin fusion protein of claim 1 wherein at least one of the immunoglobulin heavy chain CH2 constant region polypeptide and the immunoglobulin heavy chain CH3 constant region polypeptide is derived from a human immunoglobulin heavy chain.
8. The binding domain-immunoglobulin fusion protein of claim 1 wherein the immunoglobulin heavy chain constant region CH2 and CH3 polypeptides are of an isotype selected from the group consisting of human IgG and human IgA.
9. The binding domain-immunoglobulin fusion protein of claim 1 wherein the antigen is selected from the group consisting of CD19, CD20, CD37, CD40 and L6.
10. The binding domain-immunoglobulin fusion protein of claim 5 wherein the linker polypeptide comprises at least one polypeptide having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21].
11. The binding domain-immunoglobulin fusion protein of claim 5 wherein the linker polypeptide comprises at least three repeats of a polypeptide having as an amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21].
12. The binding domain-immunoglobulin fusion protein of claim 1 wherein the immunoglobulin hinge region polypeptide comprises a human IgA hinge region polypeptide.
13. The binding domain-immunoglobulin fusion protein of claim 1 wherein the binding domain polypeptide comprises a CD154 extracellular domain.
14. The binding domain-immunoglobulin fusion protein of claim 1 wherein the binding domain polypeptide comprises a CD154 extracellular domain and at least one immunoglobulin variable region polypeptide.
15. An isolated polynucleotide encoding a binding domain-immunoglobulin fusion protein, said protein comprising:
(a) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, wherein said hinge region polypeptide is selected from the group consisting of (i) a mutated hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type immunoglobulin hinge region polypeptide having one or more cysteine residues, (ii) a mutated hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type immunoglobulin hinge region polypeptide having two or more cysteine residues, (iii) a wild-type human IgA hinge region polypeptide, (iv) a mutated human IgA hinge region polypeptide that contains no cysteine residues and that is derived from a wild-type human IgA region polypeptide, and (v) a mutated human IgA hinge region polypeptide that contains one cysteine residue and that is derived from a wild-type human IgA region polypeptide;
(b) an immunoglobulin heavy chain CH2 constant region polypeptide that is fused to the hinge region polypeptide; and
(c) an immunoglobulin heavy chain CH3 constant region polypeptide that is fused to the CH2 constant region polypeptide, wherein:
(1) the binding domain-immunoglobulin fusion protein is capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity and complement fixation, and
(2) the binding domain polypeptide is capable of specifically binding to an antigen.
16. A recombinant expression construct comprising a polynucleotide according to claim 15 that is operably linked to a promoter.
17. A host cell transformed or transfected with a recombinant expression construct according to claim 16.
18. A method of producing a binding domain-immunoglobulin fusion protein, comprising the steps of:
(a) culturing a host cell according to claim 17 under conditions that permit expression of the binding domain-immunoglobulin fusion protein; and
(b) isolating the binding domain-immunoglobulin fusion protein from the host cell culture.
19. A pharmaceutical composition comprising a binding domain-immunoglobulin fusion protein according to claim 1 in combination with a physiologically acceptable carrier.
20. A method of treating a subject having or suspected of having a malignant condition or a B-cell disorder, comprising administering to a patient a therapeutically effective amount of a binding domain-immunoglobulin fusion protein according to claim 1.
21. The method of claim 20 wherein the malignant condition or B-cell disorder is selected from the group consisting of a B-cell lymphoma and a disease characterized by autoantibody production.
22. The method of claim 20 wherein the malignant condition or B-cell disorder is selected from the group consisting of rheumatoid arthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus, multiple sclerosis and an autoimmune disease.
US10053530 2001-01-17 2002-01-17 Binding domain-immunoglobulin fusion proteins Abandoned US20030133939A1 (en)

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US10053530 US20030133939A1 (en) 2001-01-17 2002-01-17 Binding domain-immunoglobulin fusion proteins
US10207655 US7754208B2 (en) 2001-01-17 2002-07-25 Binding domain-immunoglobulin fusion proteins
US10627556 US7829084B2 (en) 2001-01-17 2003-07-26 Binding constructs and methods for use thereof
PCT/US2003/024918 WO2005037989A3 (en) 2001-01-17 2003-07-26 Binding domain-immunoglobulin fusion proteins
US11089190 US20050180970A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088569 US20050202023A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088737 US20050202012A1 (en) 2001-01-17 2005-03-23 Binding domain-immunogloubulin fusion proteins
US11088693 US8106161B2 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089368 US20050238646A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088570 US20050186216A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089511 US20050175614A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089367 US20050202534A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US12371467 US20090196870A1 (en) 2001-01-17 2009-02-13 Binding constructs and methods for use thereof
US12541062 US8147835B2 (en) 2001-01-17 2009-08-13 Binding domain-immunoglobulin fusion proteins
US12724333 US20100203052A1 (en) 2001-01-17 2010-03-15 Binding domain-immunoglobulin fusion proteins
US12901297 US8197810B2 (en) 2001-01-17 2010-10-08 Binding domain-immunoglobulin fusion proteins
US12901295 US8188237B2 (en) 2001-01-17 2010-10-08 Binding domain-immunoglobulin fusion proteins
US13039097 US20110223164A1 (en) 2001-01-17 2011-03-02 Binding domain-immunoglobulin fusion proteins
US13396147 US8853366B2 (en) 2001-01-17 2012-02-14 Binding domain-immunoglobulin fusion proteins
US13451641 US9005612B2 (en) 2001-01-17 2012-04-20 Binding domain-immunoglobulin fusion proteins
US13835833 US20140010809A1 (en) 2001-01-17 2013-03-15 Binding domain-immunoglobulin fusion proteins

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US11088570 Continuation US20050186216A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088569 Continuation US20050202023A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088737 Continuation US20050202012A1 (en) 2001-01-17 2005-03-23 Binding domain-immunogloubulin fusion proteins
US11088693 Continuation US8106161B2 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089511 Continuation US20050175614A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089190 Continuation US20050180970A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089368 Continuation US20050238646A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
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US11088693 Active 2025-06-12 US8106161B2 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088569 Abandoned US20050202023A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089511 Abandoned US20050175614A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089367 Abandoned US20050202534A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089190 Abandoned US20050180970A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11089368 Abandoned US20050238646A1 (en) 2001-01-17 2005-03-23 Binding domain-immunoglobulin fusion proteins
US11088737 Abandoned US20050202012A1 (en) 2001-01-17 2005-03-23 Binding domain-immunogloubulin fusion proteins
US12901297 Active US8197810B2 (en) 2001-01-17 2010-10-08 Binding domain-immunoglobulin fusion proteins
US12901295 Active US8188237B2 (en) 2001-01-17 2010-10-08 Binding domain-immunoglobulin fusion proteins
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Cited By (220)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030118592A1 (en) * 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US20030219436A1 (en) * 2002-03-15 2003-11-27 Ledbetter Jeffrey A. Compositions and methods to regulate an immune response using CD83 gene expressed in tumors and using soluble CD83-Ig fusion protein
US20040058445A1 (en) * 2001-04-26 2004-03-25 Ledbetter Jeffrey Alan Activation of tumor-reactive lymphocytes via antibodies or genes recognizing CD3 or 4-1BB
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US20040132101A1 (en) * 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US20050069521A1 (en) * 2003-08-28 2005-03-31 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of interleukin-2 proteins
US20050123900A1 (en) * 2002-05-06 2005-06-09 Dimitrov Dimiter S. Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries
US20050164166A1 (en) * 2002-05-06 2005-07-28 Government Of The United States Of America Represented By The Secreatary, Dept Of Health Serv Identification of novel broadly cross-reactive hiv-1 neutralizing human monoclonal Antibodies
US20050164352A1 (en) * 2003-12-30 2005-07-28 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US20050202028A1 (en) * 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050244403A1 (en) * 2004-03-24 2005-11-03 Xencor, Inc. Immunoglobulin variants outside the Fc region
US20050249723A1 (en) * 2003-12-22 2005-11-10 Xencor, Inc. Fc polypeptides with novel Fc ligand binding sites
GB2416768A (en) * 2004-07-22 2006-02-08 Univ Erasmus Heavy chain immunoglobulin complexes
US20060074225A1 (en) * 2004-09-14 2006-04-06 Xencor, Inc. Monomeric immunoglobulin Fc domains
EP1648512A2 (en) * 2003-07-31 2006-04-26 Immunomedics, Inc. Anti-cd19 antibodies
US20060104971A1 (en) * 2002-12-20 2006-05-18 Biogen Idec Ma Inc. Multivalent lymphotoxin beta receptor agonists and therapeutic uses thereof
WO2006064121A2 (en) * 2004-12-15 2006-06-22 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Cytotoxic antibody directed against type b lymphoid hematopoietic proliferations
US20060173170A1 (en) * 2004-11-12 2006-08-03 Xencor, Inc. Fc variants with altered binding to FcRn
WO2006089133A2 (en) 2005-02-15 2006-08-24 Duke University Anti-cd19 antibodies and uses in oncology
US20060235208A1 (en) * 2002-09-27 2006-10-19 Xencor, Inc. Fc variants with optimized properties
US20060257403A1 (en) * 2005-04-14 2006-11-16 Young Deborah A Methods for treating and preventing fibrosis
US20060263856A1 (en) * 2001-03-07 2006-11-23 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US20060275282A1 (en) * 2005-01-12 2006-12-07 Xencor, Inc. Antibodies and Fc fusion proteins with altered immunogenicity
US20070003546A1 (en) * 2002-03-01 2007-01-04 Xencor, Inc. Optimized Fc variants and methods for their generation
US20070014776A1 (en) * 2005-06-09 2007-01-18 Gimeno Ruth E Identification of adiponutrin-related proteins as esterases and methods of use for the same
US20070020660A1 (en) * 2005-06-06 2007-01-25 Burczynski Michael E Expression profiles of peripheral blood mononuclear cells for inflammatory bowel diseases
US20070059306A1 (en) * 2005-07-25 2007-03-15 Trubion Pharmaceuticals, Inc. B-cell reduction using CD37-specific and CD20-specific binding molecules
WO2007035716A2 (en) 2005-09-16 2007-03-29 Raptor Pharmaceutical Inc. Compositions comprising receptor-associated protein (rap) variants specific for cr-containing proteins and uses thereof
US20070087000A1 (en) * 2005-08-19 2007-04-19 Walsh Frank S Antagonist antibodies against GDF-8 and uses in treatment of ALS and other GDF-8-associated disorders
US20070148171A1 (en) * 2002-09-27 2007-06-28 Xencor, Inc. Optimized anti-CD30 antibodies
US20070154473A1 (en) * 2005-12-30 2007-07-05 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US20070154453A1 (en) * 2005-12-30 2007-07-05 Merck Patent Gmbh Interleukin-12p40 variants with improved stability
US20070212349A1 (en) * 2001-10-16 2007-09-13 Government Of The United States Of America, Represented By The Secretary, Department Broadly cross-reactive neutralizing antibodies against human immunodeficiency virus selected by env-cd4-co-receptor complexes
US20070219133A1 (en) * 2002-03-01 2007-09-20 Xencor, Inc. CD52 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
US20070238665A1 (en) * 2003-03-03 2007-10-11 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIc
JP2007528194A (en) * 2003-07-26 2007-10-11 トルビオン・ファーマシューティカルズ・インコーポレイテッドTrubion Pharmaceuticals, Inc. Binding constructs and methods of use thereof
US20070253950A1 (en) * 2006-03-21 2007-11-01 Wyeth Methods for Preventing and Treating Amyloidogenic Diseases
US20070274987A1 (en) * 2006-05-25 2007-11-29 Valerie Clerin Expression of the cysteine protease legumain in vascular and inflammatory diseases
US20070275460A1 (en) * 2003-03-03 2007-11-29 Xencor.Inc. Fc Variants With Optimized Fc Receptor Binding Properties
US20070287160A1 (en) * 2006-04-21 2007-12-13 Chou Judy H Methods for high throughput screening of cell lines
US20070292390A1 (en) * 2004-10-29 2007-12-20 Dimitrov Dimiter S Broadly Cross-Reactive Hiv-1 Neutralizing Human Monoclonal Antibodies
US20080027001A1 (en) * 2006-07-07 2008-01-31 Andrew Wood Nogo receptor functional motifs, peptide mimetics, and mutated functional motifs related thereto, and methods of using the same
US20080038280A1 (en) * 2004-10-14 2008-02-14 Government Of The United States Of America A32 Monoclonal Antibody Fusion Proteins For Use As Hiv Inhibitors And Vaccines
US20080038258A1 (en) * 2006-07-21 2008-02-14 Amgen Inc. Polypeptides with Reduced Susceptibility to Oxidation and Methods of Making
US20080057056A1 (en) * 2003-03-03 2008-03-06 Xencor, Inc. Fc Variants with Increased Affinity for FcyRIIC
US20080064861A1 (en) * 2006-09-08 2008-03-13 Wyeth Arginine wash in protein purification using affinity chromotography
US20080131431A1 (en) * 2006-05-15 2008-06-05 Viral Logic Systems Technology Corp. CD47 related compositions and methods for treating immunological diseases and disorders
US20080132688A1 (en) * 2006-09-22 2008-06-05 Amgen Inc. Methods for Removing Viral Contaminants During Protein Purification
US20080182895A1 (en) * 2006-08-25 2008-07-31 Howe Anita Y M Identification and characterization of hcv replicon variants with reduced susceptibility to hcv-796, and methods related thereto
US20080206867A1 (en) * 2005-10-03 2008-08-28 Desjarlais John R Fc variants with optimized Fc receptor binding properties
US20080213277A1 (en) * 2007-02-02 2008-09-04 Amgen Inc. Hepcidin, hepcidin antagonists and methods of use
US20080241130A1 (en) * 2007-03-28 2008-10-02 Wright Jill F Methods and compositions for modulating il-17f/il-17a biological activity
US20080254027A1 (en) * 2002-03-01 2008-10-16 Bernett Matthew J Optimized CD5 antibodies and methods of using the same
US20080260731A1 (en) * 2002-03-01 2008-10-23 Bernett Matthew J Optimized antibodies that target cd19
US20080269132A1 (en) * 2007-04-23 2008-10-30 Wyeth Use of low temperature and/or low ph in cell culture
US20080267976A1 (en) * 2005-10-06 2008-10-30 Gregory Alan Lazar Optimized Anti-Cd30 Antibodies
US20080279850A1 (en) * 2005-07-25 2008-11-13 Trubion Pharmaceuticals, Inc. B-Cell Reduction Using CD37-Specific and CD20-Specific Binding Molecules
WO2008145338A2 (en) 2007-05-29 2008-12-04 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US20080313379A1 (en) * 2007-06-15 2008-12-18 United Memories, Inc. Multiple bus charge sharing
US20090041765A1 (en) * 2006-10-24 2009-02-12 Trubion Pharmaceuticals, Inc. Materials and methods for improved immunoglycoproteins
US20090042253A1 (en) * 2007-08-09 2009-02-12 Wyeth Use of perfusion to enhance production of fed-batch cell culture in bioreactors
US20090131327A1 (en) * 2005-04-29 2009-05-21 Patrick Doherty Nogo receptor functional motifs and peptide mimetics related thereto and methods of using the same
EP2062591A1 (en) 2005-04-07 2009-05-27 Novartis Vaccines and Diagnostics, Inc. CACNA1E in cancer diagnosis detection and treatment
US20090142806A1 (en) * 2005-02-14 2009-06-04 Wyeth Interleukin-17f antibodies and other il-17f signaling antagonists and uses therefor
US20090142261A1 (en) * 2006-08-04 2009-06-04 Novartis Ag EPHB3-Specific Antibody and Uses Thereof
US20090148436A1 (en) * 2007-11-01 2009-06-11 Wyeth Antibody to gdf8 and uses thereof
WO2009086072A2 (en) 2007-12-21 2009-07-09 Genentech, Inc. Therapy of rituximab-refractory rheumatoid arthritis patients
WO2009085200A2 (en) 2007-12-21 2009-07-09 Amgen Inc. Anti-amyloid antibodies and uses thereof
WO2009086003A1 (en) 2007-12-20 2009-07-09 Xoma Technology Ltd. Methods for the treatment of gout
EP2083088A2 (en) 2005-04-07 2009-07-29 Novartis Vaccines and Diagnostics, Inc. Cancer-related genes
WO2009094551A1 (en) 2008-01-25 2009-07-30 Amgen Inc. Ferroportin antibodies and methods of use
WO2009126944A1 (en) 2008-04-11 2009-10-15 Trubion Pharmaceuticals, Inc. Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
US20090258005A1 (en) * 2007-05-29 2009-10-15 Trubion Pharmaceuticals Inc. Therapeutic compositions and methods
WO2009139822A1 (en) 2008-05-01 2009-11-19 Amgen Inc. Anti-hepcidin antibodies and methods of use
US20090304590A1 (en) * 2007-05-29 2009-12-10 Wyeth Therapeutic compositions and methods
US20100040614A1 (en) * 2006-12-27 2010-02-18 Rafi Ahmed Compositions and methods for the treatment of infections and tumors
EP2163562A2 (en) 2005-06-21 2010-03-17 XOMA Technology Ltd. IL-1beta binding antibodies and fragments thereof
EP2166021A1 (en) 2008-09-16 2010-03-24 Ganymed Pharmaceuticals AG Monoclonal antibodies for treatment of cancer
US7704508B2 (en) 2007-09-14 2010-04-27 New York Blood Center Babesia subtilisin
US7704503B2 (en) 2005-02-14 2010-04-27 Wyeth Llc Use of IL-17F in diagnosis and therapy of airway inflammation
US20100104557A1 (en) * 2006-09-18 2010-04-29 Xencor, Inc. Optimized Antibodies that Target HM1.24
US20100113304A1 (en) * 2008-09-26 2010-05-06 Wyeth Compatible display vector systems
US20100111975A1 (en) * 2007-03-14 2010-05-06 Ugur Sahin Monoclonal Antibodies for Treatment of Cancer
WO2010056804A1 (en) 2008-11-12 2010-05-20 Medimmune, Llc Antibody formulation
US20100135900A1 (en) * 2008-11-13 2010-06-03 Trubion Pharmaceuticals, Inc. Cd37 immunotherapeutic combination therapies and uses thereof
US20100150948A1 (en) * 2006-10-24 2010-06-17 Trubion Pharmaceuticals, Inc. Materials and methods for improved immunoglycoproteins
US20100151492A1 (en) * 2008-11-28 2010-06-17 Emory University And Dana Farber Cancer Institute, Inc. Methods for the treatment of infections and tumors
WO2010075249A2 (en) 2008-12-22 2010-07-01 Genentech, Inc. A method for treating rheumatoid arthritis with b-cell antagonists
US20100189722A1 (en) * 2007-08-09 2010-07-29 Boehringer Ingelheim International Gmbh Anti cd37 antibodies
EP2221316A1 (en) 2005-05-05 2010-08-25 Duke University Anti-CD19 antibody therapy for autoimmune disease
WO2010096813A1 (en) 2009-02-23 2010-08-26 New York Blood Center Krüppel-like factors and fat regulation
WO2010105128A2 (en) 2009-03-12 2010-09-16 Wyeth Llc A pkn3/rhoc macromolecular complex and methods of use therefor
WO2010102886A1 (en) 2009-02-19 2010-09-16 Novo Nordisk A/S Modification of factor viii
US20100239579A1 (en) * 2006-05-15 2010-09-23 Viral Logic Systems Technology Corp. CD47 Related Compositions and Methods for Treating Immunological Diseases and Disorders
WO2010111378A1 (en) 2009-03-24 2010-09-30 Wyeth Llc Membrane evaporation for generating highly concentrated protein therapeutics
WO2010112034A2 (en) 2009-04-02 2010-10-07 Aarhus Universitet Compositions and methods for treatment and diagnosis of synucleinopathies
WO2010114860A1 (en) 2009-03-30 2010-10-07 Acceleron Pharma Inc. Bmp-alk3 antagonists and uses for promoting bone growth
US20100272723A1 (en) * 2006-08-14 2010-10-28 Xencor, Inc. Optimized Antibodies that Target CD19
US20100297106A1 (en) * 2007-09-27 2010-11-25 Christopher James Sloey Pharmaceutical Formulations
US20100310646A1 (en) * 2008-01-25 2010-12-09 Claus Oxvig Selective exosite inhibition of papp-a activity against igfbp-4
WO2010147686A2 (en) 2009-03-11 2010-12-23 Wyeth Llc Methods of purifying small modular immunopharmaceutical proteins
WO2011003996A1 (en) 2009-07-09 2011-01-13 F. Hoffmann-La Roche Ag In vivo tumor vasculature imaging
US20110014624A1 (en) * 2008-03-12 2011-01-20 Wyeth Llc Methods For Identifying Cells Suitable For Large-Scale Production of Recombinant Proteins
EP2295469A2 (en) 2005-11-24 2011-03-16 Ganymed Pharmaceuticals AG Monoclonal antibodies against claudin-18 for treatment of cancer
WO2011038301A2 (en) 2009-09-25 2011-03-31 Xoma Technology Ltd. Screening methods
WO2011038302A2 (en) 2009-09-25 2011-03-31 Xoma Technology Ltd. Novel modulators
US20110081345A1 (en) * 2007-04-18 2011-04-07 Moore Margaret D Single chain fc, methods of making and methods of treatment
EP2322555A1 (en) 2009-11-11 2011-05-18 Ganymed Pharmaceuticals AG Antibodies specific for claudin 6 (CLDN6)
WO2011057788A1 (en) 2009-11-11 2011-05-19 Ganymed Pharmaceuticals Ag Antibodies specific for claudin 6 (cldn6)
WO2011063277A1 (en) 2009-11-20 2011-05-26 Amgen Inc. Anti-orai1 antigen binding proteins and uses thereof
EP2327423A2 (en) 2006-02-21 2011-06-01 Wyeth LLC Human antibodies against human interleukin-22 (IL-22)
WO2011063980A1 (en) 2009-11-26 2011-06-03 Inflarx Gmbh Anti-c5a binding moieties with high blocking activity
EP2329837A1 (en) 2005-11-23 2011-06-08 Acceleron Pharma Inc. Promoting bone growth
US20110142851A1 (en) * 2008-07-02 2011-06-16 Lynda Misher IL6 Immunotherapeutics
US20110150760A1 (en) * 2006-08-18 2011-06-23 Novartis Ag PRLR-Specific Antibody and Uses Thereof
WO2011079308A2 (en) 2009-12-23 2011-06-30 Emergent Product Development Seattle, Llc Compositions comprising tnf-alpha and il-6 antagonists and methods of use thereof
WO2011090761A1 (en) 2009-12-29 2011-07-28 Emergent Product Development Seattle, Llc Ron binding constructs and methods of use thereof
US20110189093A1 (en) * 2008-04-14 2011-08-04 Proscan Rx Pharma Prostate specific membrane antigen antibodies and antigen binding fragments
US20110189179A1 (en) * 2005-09-12 2011-08-04 Ugur Sahin Identification of Tumor-Associated Antigens for Diagnosis and Therapy
WO2011100403A1 (en) 2010-02-10 2011-08-18 Immunogen, Inc Cd20 antibodies and uses thereof
US20110217302A1 (en) * 2008-10-10 2011-09-08 Emergent Product Development Seattle, Llc TCR Complex Immunotherapeutics
WO2011116885A1 (en) 2010-03-23 2011-09-29 Ganymed Pharmaceuticals Ag Monoclonal antibodies for treatment of cancer
WO2011125012A1 (en) 2010-04-05 2011-10-13 Wyeth Llc Biomarkers for p13k-driven cancer
WO2011131510A2 (en) 2010-04-21 2011-10-27 Novo Nordisk A/S Selective modification of proteins
WO2011143307A1 (en) 2010-05-14 2011-11-17 Amgen Inc. High concentration antibody formulations
WO2011157275A1 (en) 2010-06-14 2011-12-22 H. Lundbeck A/S Modulation of the interaction between sorla and gdnf-family ligand receptors
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
EP2404936A1 (en) 2010-07-06 2012-01-11 Ganymed Pharmaceuticals AG Cancer therapy using CLDN6 target-directed antibodies in vivo
EP2407483A1 (en) 2006-04-13 2012-01-18 Novartis Vaccines and Diagnostics, Inc. Methods of treating, diagnosing or detecting cancers
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
WO2012014183A1 (en) 2010-07-30 2012-02-02 Pfizer Inc. Tandem purification of proteins
EP2415483A1 (en) 2005-07-25 2012-02-08 Emergent Product Development Seattle, LLC Single dose use of cd20-specific binding molecules
EP2418223A2 (en) 2006-06-12 2012-02-15 Emergent Product Development Seattle, LLC Single-chain multivalent binding proteins with effector function
WO2012028622A2 (en) 2010-08-31 2012-03-08 Sanofi Peptide or peptide complex binding to 2 integrin and methods and uses involving the same
EP2431392A1 (en) 2006-02-21 2012-03-21 Wyeth LLC Antibodies against human IL-22 and uses therefor
WO2012040518A2 (en) 2010-09-22 2012-03-29 Amgen Inc. Carrier immunoglobulins and uses thereof
US8188231B2 (en) 2002-09-27 2012-05-29 Xencor, Inc. Optimized FC variants
EP2474556A2 (en) 2007-03-14 2012-07-11 Novartis AG APCDD1 inhibitors for treating, diagnosing or detecting cancer
EP2481758A1 (en) 2011-01-28 2012-08-01 Sanofi Human antibodies to PSCK9 for use in methods of treating particular groups of subjects (11566)
WO2012101252A2 (en) 2011-01-28 2012-08-02 Sanofi Human antibodies to pcsk9 for use in methods of treating particular groups of subjects
EP2494988A1 (en) 2006-12-07 2012-09-05 Novartis AG Antagonist antibodies against EPHB3
WO2012125459A1 (en) 2011-03-11 2012-09-20 Celgene Corporation Methods of treating cancer using 3-(5-amino-2-methyl-4-oxo-4h-quinazolin-3-yl)-piperidine-2,6-dione
US8278421B2 (en) 2006-03-20 2012-10-02 Xoma Techolology Ltd. Human antibodies specific for gastrin materials and methods
WO2012146776A1 (en) 2011-04-29 2012-11-01 Sanofi Test systems and methods for identifying and characterising lipid lowering drugs
US8313942B2 (en) 2008-09-26 2012-11-20 Wyeth Llc Compatible display vector systems
WO2012156018A1 (en) 2011-05-13 2012-11-22 Ganymed Pharmaceuticals Ag Antibodies for treatment of cancer expressing claudin 6
US8318907B2 (en) 2004-11-12 2012-11-27 Xencor, Inc. Fc variants with altered binding to FcRn
WO2012162561A2 (en) 2011-05-24 2012-11-29 Zyngenia, Inc. Multivalent and monovalent multispecific complexes and their uses
EP2535355A2 (en) 2005-03-23 2012-12-19 Genmab A/S Antibodies against CD38 for treatment of multiple myeloma
US8337840B2 (en) 2003-07-31 2012-12-25 Immunomedics, Inc. Anti-CD19 antibodies
EP2540741A1 (en) 2006-03-06 2013-01-02 Aeres Biomedical Limited Humanized anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease
US8426562B2 (en) 2005-09-13 2013-04-23 National Research Council Of Canada Methods and compositions for modulating tumor cell activity
US8431126B2 (en) 2006-02-13 2013-04-30 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
WO2013096516A1 (en) 2011-12-19 2013-06-27 Xoma Technology Ltd. Methods for treating acne
US20130177568A1 (en) * 2012-01-09 2013-07-11 Icb International, Inc. Blood-brain barrier permeable peptide compositions
US8546543B2 (en) 2004-11-12 2013-10-01 Xencor, Inc. Fc variants that extend antibody half-life
EP2650016A1 (en) 2011-01-28 2013-10-16 Sanofi Human antibodies to PSCK9 for use in methods of treatment based on particular dosage regimens (11565)
US8569462B2 (en) 2011-06-03 2013-10-29 Xoma Technology Ltd. Antibodies specific for TGF-beta and methods for treating thereof
US8580257B2 (en) 2008-11-03 2013-11-12 Alethia Biotherapeutics Inc. Antibodies that specifically block the biological activity of kidney associated antigen 1 (KAAG1)
EP2662386A1 (en) 2012-05-07 2013-11-13 BioNTech AG Antibodies that bind to PLAC1 and block interaction between PLAC1 and FGF7
WO2013167153A1 (en) 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Antibodies useful in cancer diagnosis
WO2013174404A1 (en) 2012-05-23 2013-11-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2013174403A1 (en) 2012-05-23 2013-11-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2013185117A1 (en) 2012-06-07 2013-12-12 Ambrx, Inc. Prostate-specific membrane antigen antibody drug conjugates
WO2013188740A1 (en) 2012-06-14 2013-12-19 Ambrx, Inc. Anti-psma antibodies conjugated to nuclear receptor ligand polypeptides
WO2013192360A1 (en) 2012-06-19 2013-12-27 Ambrx, Inc. Anti-cd70 antibody drug conjugates
WO2014025960A1 (en) 2012-08-09 2014-02-13 Celgene Corporation Methods of treating cancer using 3-(4-((4-(morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
US8652465B2 (en) 2005-06-08 2014-02-18 Emory University Methods and compositions for the treatment of persistent infections
EP2703008A1 (en) 2012-08-31 2014-03-05 Sanofi Human antibodies to PCSK9 for use in methods of treating particular groups of subjects
EP2703011A2 (en) 2007-05-07 2014-03-05 MedImmune, LLC Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease
EP2703009A1 (en) 2012-08-31 2014-03-05 Sanofi Combination treatments involving antibodies to human PCSK9
EP2706070A1 (en) 2012-09-06 2014-03-12 Sanofi Combination treatments involving antibodies to human PCSK9
WO2014046994A1 (en) 2012-09-18 2014-03-27 University Of Washington Through Its Center For Commercialization Compositions and methods for antigen targeting to cd180
WO2014047124A1 (en) 2012-09-18 2014-03-27 University Of Washington Through Its Center For Commercialization Compositions and methods for delivery of antigens to plasmacytoid dendritic cells
EP2732823A1 (en) 2008-06-25 2014-05-21 H. Lundbeck A/S Modulation of the TrpV : Vps10p-domain receptor system for the treatment of pain
WO2014075697A1 (en) 2012-11-13 2014-05-22 Biontech Ag Agents for treatment of claudin expressing cancer diseases
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
US20140161800A1 (en) * 2011-04-22 2014-06-12 John W. Blankenship Prostate-Specific Membrane Antigen Binding Proteins and Related Compositions and Methods
US8802820B2 (en) 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
US8802826B2 (en) 2009-11-24 2014-08-12 Alethia Biotherapeutics Inc. Anti-clusterin antibodies and antigen binding fragments and their use to reduce tumor volume
US8802685B2 (en) 2011-03-11 2014-08-12 Celgene Corporation Solid forms of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, and their pharmaceutical compositions and uses
WO2014127785A1 (en) 2013-02-20 2014-08-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2014146672A1 (en) 2013-03-18 2014-09-25 Ganymed Pharmaceuticals Ag Therapy involving antibodies against claudin 18.2 for treatment of cancer
US8937163B2 (en) 2011-03-31 2015-01-20 Alethia Biotherapeutics Inc. Antibodies against kidney associated antigen 1 and antigen binding fragments thereof
WO2015028027A1 (en) 2013-08-29 2015-03-05 University Of Copenhagen Anti-ADAM12 antibodies for the treatment of cancer
US8992915B2 (en) 2012-05-16 2015-03-31 Boehringer Ingelheim International Gmbh Combination of CD37 antibodies with ICE
US8992913B2 (en) 2012-06-15 2015-03-31 Pfizer Inc. Antagonist antibodies against GDF-8 and uses therefor
EP2862579A1 (en) 2006-10-04 2015-04-22 Dana-Farber Cancer Institute, Inc. Tumor immunity
US9051373B2 (en) 2003-05-02 2015-06-09 Xencor, Inc. Optimized Fc variants
WO2015113576A1 (en) 2014-01-29 2015-08-06 Biontech Ag Peptide mimotopes of claudin 18.2 and uses thereof
EP2910645A1 (en) 2014-02-25 2015-08-26 STRATIFYER Molecular Pathology GmbH Methods and compositions for prediction of therapeutic efficacy of cancer treatments and cancer prognosis upon antibody treatment
WO2015150327A1 (en) 2014-04-01 2015-10-08 Biontech Cell & Gene Therapies Gmbh Claudin-6-specific immunoreceptors and t cell epitopes
US9193794B2 (en) 2006-06-07 2015-11-24 Bioalliance C.V. Antibodies recognizing a carbohydrate containing epitope on CD-43 and CEA expressed on cancer cells and methods using same
WO2015179627A1 (en) 2014-05-21 2015-11-26 Dana-Farber Cancer Institute, Inc. Methods for treating cancer with anti bip or anti mica antibodies
US9260529B2 (en) 2010-02-24 2016-02-16 The University Of Washington Through Its Center For Commercialization Molecules that bind CD180, compositions and methods of use
WO2016044224A1 (en) 2014-09-15 2016-03-24 Amgen Inc. Bi-specific anti-cgrp receptor/pac1 receptor antigen binding proteins and uses thereof
US9334329B2 (en) 2007-12-18 2016-05-10 Bioalliance C.V. Antibodies recognizing a carbohydrate containing epitope on CD-43 and CEA expressed on cancer cells and methods using same
WO2016141111A1 (en) 2015-03-03 2016-09-09 Xoma (Us) Llc Treatment of post-prandial hyperinsulinemia and hypoglycemia after bariatric surgery
WO2016146134A1 (en) 2015-03-16 2016-09-22 Aarhus Universitet Antibodies towards an extracellular region of nbcn1
WO2016161410A2 (en) 2015-04-03 2016-10-06 Xoma Technology Ltd. Treatment of cancer using inhibitors of tgf-beta and pd-1
WO2016166124A1 (en) 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Methods and compositions for prediction of therapeutic efficacy of cancer treatments and cancer prognosis
WO2016166122A1 (en) 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Drug conjugates comprising antibodies against claudin 18.2
US9475881B2 (en) 2010-01-19 2016-10-25 Xencor, Inc. Antibody variants with enhanced complement activity
US9493564B2 (en) 2008-10-02 2016-11-15 Aptevo Research And Development Llc CD86 antagonist multi-target binding proteins
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies
WO2016180468A1 (en) 2015-05-11 2016-11-17 Biontech Cell & Gene Therapies Gmbh Claudin-18.2-specific immunoreceptors and t cell epitopes
WO2016180778A1 (en) 2015-05-11 2016-11-17 Biontech Cell & Gene Therapies Gmbh Enhancing the effect of car-engineered t cells by means of nucleic acid vaccination
EP3095463A2 (en) 2008-09-16 2016-11-23 F. Hoffmann-La Roche AG Methods for treating progressive multiple sclerosis
US9550837B2 (en) 2008-12-15 2017-01-24 Regeneron Pharmaceuticals, Inc. Therapeutic uses of anti-PCSK9 antibodies
EP3124045A2 (en) 2006-12-20 2017-02-01 Xoma (Us) Llc Treatment of il-1 beta related diseases
WO2017049004A1 (en) 2015-09-15 2017-03-23 Amgen Inc. Tetravalent bispecific and tetraspecific antigen binding proteins and uses thereof
WO2017060300A1 (en) 2015-10-07 2017-04-13 Biontech Cell & Gene Therapies Gmbh Antigen receptors and uses thereof
US9657098B2 (en) 2013-03-15 2017-05-23 Intrinsic Lifesciences, Llc Anti-hepcidin antibodies and uses thereof
WO2017106578A1 (en) 2015-12-15 2017-06-22 Amgen Inc. Pacap antibodies and uses thereof
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
US9724411B2 (en) 2008-12-15 2017-08-08 Regeneron Pharmaceuticals, Inc. Methods for treating hypercholesterolemia and reducing LDL-C using antibodies to PCSK9
US9775785B2 (en) 2004-05-18 2017-10-03 Ganymed Pharmaceuticals Ag Antibody to genetic products differentially expressed in tumors and the use thereof
US9809815B2 (en) 2009-02-20 2017-11-07 Ganymed Pharmaceuticals Ag Methods and compositions for diagnosis and treatment of cancer
US9822170B2 (en) 2012-02-22 2017-11-21 Alethia Biotherapeutics Inc. Co-use of a clusterin inhibitor with an EGFR inhibitor to treat cancer
US9873745B2 (en) 2004-12-15 2018-01-23 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Antibody method for treatment of a disease in which the target cells are cells which express CD20

Families Citing this family (126)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69942782D1 (en) * 1998-11-27 2010-10-28 Ucb Sa Compositions and methods for increasing bone mineralization
US20040009535A1 (en) 1998-11-27 2004-01-15 Celltech R&D, Inc. Compositions and methods for increasing bone mineralization
US20050281821A1 (en) * 1999-01-06 2005-12-22 Flavia Pernasetti Method and composition for angiogenesis inhibition
CA2447851C (en) * 2001-06-28 2012-08-28 Domantis Limited Dual-specific ligand and its use
ES2263984T3 (en) * 2002-06-28 2006-12-16 Domantis Limited Double-specific ligands with increased serum half-life.
US9321832B2 (en) * 2002-06-28 2016-04-26 Domantis Limited Ligand
US7696320B2 (en) 2004-08-24 2010-04-13 Domantis Limited Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
CN1642983A (en) 2002-01-25 2005-07-20 G2治疗有限公司 Antibodies against of C5aR and use thereof
WO2003087763A3 (en) 2002-04-03 2007-09-07 Celltech R & D Inc Association of polymorphisms in the sost gene region with bone mineral density
EP1578801A2 (en) * 2002-12-27 2005-09-28 Domantis Limited Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
CA2529623A1 (en) 2003-06-16 2005-02-17 Celltech R & D, Inc. Antibodies specific for sclerostin and methods for increasing bone mineralization
US7935790B2 (en) * 2004-10-04 2011-05-03 Cell Singaling Technology, Inc. Reagents for the detection of protein phosphorylation in T-cell receptor signaling pathways
EP1776384B1 (en) 2004-08-04 2013-06-05 Mentrik Biotech, LLC Variant fc regions
US20060110387A1 (en) * 2004-10-05 2006-05-25 Genentech, Inc. Method for treating vasculitis
DE602006002305D1 (en) * 2005-04-22 2008-10-02 Effebi Spa Connection plate between an armature and a drive
US7592429B2 (en) 2005-05-03 2009-09-22 Ucb Sa Sclerostin-binding antibody
US8003108B2 (en) 2005-05-03 2011-08-23 Amgen Inc. Sclerostin epitopes
RU2007147426A (en) * 2005-05-20 2009-06-27 Дженентек, Инк. (Us) Pre-treatment of a biological sample taken from an individual with an autoimmune disease
WO2007021776A3 (en) * 2005-08-09 2009-04-23 Univ Texas Methods and devices for breast reconstruction
WO2007024921A3 (en) * 2005-08-24 2007-06-14 Peter C Brooks Combination therapies for inhibiting integrin-extracellular matrix interactions
ES2611307T3 (en) 2005-08-30 2017-05-08 University Of Miami Immunomodulation of agonists, antagonists and immunotoxins receptor tumor necrosis factor 25 (TNFR25)
CN101454344A (en) * 2005-12-01 2009-06-10 杜门蒂斯有限公司 Noncompetitive domain antibody formats that bind interleukin 1 receptor type 1
US20080107597A1 (en) * 2006-01-12 2008-05-08 Anaptys Biosciences, Inc. Isolation of antibodies that cross-react and neutralize rankl originating from multiple species
US7625564B2 (en) * 2006-01-27 2009-12-01 Novagen Holding Corporation Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
EP2359834B1 (en) 2006-03-15 2016-11-09 Alexion Pharmaceuticals, Inc. Treatment of paroxysmal nocturnal hemoglobinuria patients by an inhibitor of complement
EP2038306B1 (en) 2006-06-30 2014-12-03 Novo Nordisk A/S Anti-nkg2a antibodies and uses thereof
EP1983002A3 (en) 2007-04-19 2009-03-11 Peter Hornbeck Tyrosine phosphorylation sites and antibodies specific for them
US9388235B2 (en) 2006-09-05 2016-07-12 Alexion Pharmaceuticals, Inc. Methods and compositions for the treatment of antibody mediated neuropathies
EP2064239B1 (en) 2006-09-08 2014-11-26 Amgen Inc. Anti-activin a antibodies and uses thereof
EP2423226A3 (en) 2006-11-10 2012-05-30 Amgen Inc. Antibody-based diagnostics and therapeutics
EP2460828A3 (en) * 2006-11-10 2012-08-08 UCB Pharma, S.A. Antibodies and diagnostics
KR101431856B1 (en) 2007-01-22 2014-08-27 제넨테크, 인크. Polyelectrolyte precipitation and purification of antibodies
EP2132573B1 (en) 2007-03-02 2014-04-23 Genentech, Inc. Predicting response to a her dimerisation inhbitor based on low her3 expression
US20090081659A1 (en) 2007-03-07 2009-03-26 Cell Signaling Technology, Inc. Reagents for the detection of protein phosphorylation in carcinoma signaling pathways
EP1975184A3 (en) 2007-03-26 2008-11-26 Cell Signaling Technology, Inc. Serine or threonine phosphorylation sites
EP1983003A3 (en) 2007-04-19 2009-03-11 Peter Hornbeck Tyrosine phosphorylation sites and antibodies specific for them
US20090053831A1 (en) 2007-05-01 2009-02-26 Cell Signaling Technology, Inc. Tyrosine phosphorylation sites
EP2155238B1 (en) 2007-06-05 2016-04-06 Yale University Antibody against d4 domain of the kit receptor and use thereor
WO2008149147A3 (en) 2007-06-06 2009-06-11 Domantis Ltd Polypeptides, antibody variable domains and antagonists
WO2009023386A3 (en) * 2007-07-06 2009-11-19 Trubion Pharmaceuticals, Inc. Binding peptides having a c-terminally disposed specific binding domain
WO2009012600A1 (en) * 2007-07-26 2009-01-29 Novagen Holding Corporation Fusion proteins
CN105330743A (en) 2007-08-02 2016-02-17 吉联亚生物科技有限公司 LOX and LOXL2 inhibitors and uses thereof
WO2009023185A1 (en) 2007-08-13 2009-02-19 Vasgene Therapeutics, Inc. Cancer treatment using humanized antibodies that bind to ephb4
JP5643094B2 (en) 2007-09-17 2014-12-17 アムジエン・インコーポレーテツド Method of inhibiting bone resorption
EP2227556B1 (en) 2007-11-19 2014-08-27 Celera Corporation Lung cancer markers and uses thereof
JP5615181B2 (en) 2007-11-27 2014-10-29 ザ・ユニバーシティ・オブ・ブリティッシュ・コロンビア Compositions and methods for the prevention and treatment of arthritis
KR101615935B1 (en) 2007-12-14 2016-04-28 노보 노르디스크 에이/에스 Antibodies against human nkg2d and uses thereof
EP2628486A3 (en) 2007-12-14 2013-10-30 Amgen, Inc. Method for treating bone fracture with anti-sclerostin antibodies
EP2231181B1 (en) 2007-12-17 2016-02-17 Marfl AB New vaccine for the treatment of mycobacterium related disorders
CN101952317B (en) 2008-01-24 2015-07-22 诺沃-诺迪斯克有限公司 Humanized anti-human NKG2A monoclonal antibody
CN101970494B (en) 2008-02-20 2015-01-21 G2炎症私人有限公司 Humanized anti-c5ar antibodies
US20090220991A1 (en) * 2008-02-29 2009-09-03 Cell Signaling Technology, Inc. Reagents for the detection of protein phosphorylation in leukemia signaling pathways
WO2009126306A3 (en) 2008-04-10 2010-04-22 Cell Signaling Technology, Inc. Compositions and methods for detecting egfr mutations in cancer
US20090269339A1 (en) * 2008-04-29 2009-10-29 Genentech, Inc. Responses to immunizations in rheumatoid arthritis patients treated with a cd20 antibody
US8148088B2 (en) * 2008-07-18 2012-04-03 Abgent Regulation of autophagy pathway phosphorylation and uses thereof
US20100082438A1 (en) * 2008-10-01 2010-04-01 Ronnie Jack Garmon Methods and systems for customer performance scoring
WO2010042433A1 (en) 2008-10-06 2010-04-15 Bristol-Myers Squibb Company Combination of cd137 antibody and ctla-4 antibody for the treatment of proliferative diseases
JP5714505B2 (en) 2008-12-22 2015-05-07 ノヴォ ノルディスク アー/エス Antibody to tissue factor pathway inhibitor
ES2539045T3 (en) 2009-01-19 2015-06-25 Innate Pharma Anti-KIR3D
EP2448593A2 (en) 2009-07-03 2012-05-09 Bionor Immuno AS Hiv related peptides combination or fusion for use in hiv vaccine composition or as diagnostic means
WO2011017303A1 (en) 2009-08-03 2011-02-10 University Of Miami Method for in vivo expansion of t regulatory cells
US9493578B2 (en) 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
US8221753B2 (en) 2009-09-30 2012-07-17 Tracon Pharmaceuticals, Inc. Endoglin antibodies
ES2626000T3 (en) 2009-11-02 2017-07-21 University Of Washington Therapeutic compositions and methods of nucleases
US20120282276A1 (en) 2009-11-05 2012-11-08 The Regents Of The University Of Michigan Biomarkers predictive of progression of fibrosis
WO2011075636A3 (en) 2009-12-18 2011-10-20 Amgen Inc. Wise binding agents and epitopes
US20120296403A1 (en) 2010-02-10 2012-11-22 Novartis Ag Methods and compounds for muscle growth
WO2011146568A8 (en) 2010-05-19 2012-06-28 F. Hoffmann-La Roche Ag Predicting response to a her inhibitor
WO2011159826A3 (en) 2010-06-15 2013-07-04 Vertex Pharmaceuticals Incorporated Hcv ns5b polymerase mutants
WO2012000994A1 (en) 2010-06-29 2012-01-05 C.N.R.S. Llt-1 antibodies with new functional properties
JP2013533871A (en) 2010-06-30 2013-08-29 ノヴォ ノルディスク アー/エス Capable of specifically binding to tissue factor pathway inhibitor antibodies
JP5953303B2 (en) 2010-07-29 2016-07-20 ゼンコア インコーポレイテッド Antibodies having isoelectric points that have been modified
CA2806855A1 (en) 2010-08-03 2012-02-09 F. Hoffmann - La Roche Ag Chronic lymphocytic leukemia (cll) biomarkers
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
US9079958B2 (en) 2010-10-04 2015-07-14 Boehringer Ingelheim International Gmbh CD33 binding agents
US20140308242A1 (en) 2010-10-21 2014-10-16 Vertex Pharmaceuticals Incorporated Biomarkers for hcv infected patients
KR20130086628A (en) 2010-11-08 2013-08-02 제넨테크, 인크. Subcutaneously administered anti-il-6 receptor antibody
US20120171120A1 (en) 2010-11-30 2012-07-05 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses therefor
EP2681242B1 (en) 2011-03-01 2018-01-24 Amgen Inc. Sclerostin and dkk-1 bispecific binding agents
US9133270B2 (en) * 2011-03-08 2015-09-15 The Trustees Of The University Of Pennsylvania Antibody-like proteins for therapeutic and diagnostic use
CA2830538A1 (en) 2011-03-25 2012-10-04 Amgen Inc. Anti - sclerostin antibody crystals and formulations thereof
WO2012145417A1 (en) 2011-04-19 2012-10-26 Amgen Inc. Method for treating osteoporosis
CA2834626A1 (en) 2011-04-29 2012-11-01 University Of Washington Therapeutic nuclease compositions and methods
WO2012154983A3 (en) 2011-05-10 2013-04-11 Biocare Medical, Llc Systems and methods for anti-pax8 antibodies
WO2012164021A1 (en) 2011-05-31 2012-12-06 Novo Nordisk A/S Il-21 epitope and il-21 ligands
EP2718322A1 (en) 2011-06-06 2014-04-16 Novo Nordisk A/S Therapeutic antibodies
US9512228B2 (en) 2011-06-17 2016-12-06 Novo Nordisk A/S Selective elimination of erosive cells
WO2013011059A1 (en) 2011-07-18 2013-01-24 Novo Nordisk A/S Antagonist antibodies against oscar
WO2013011062A3 (en) 2011-07-18 2013-03-21 Novo Nordisk A/S Antagonistic antibodies against oscar
WO2013011063A1 (en) 2011-07-18 2013-01-24 Novo Nordisk A/S Antagonistic antibodies against oscar
WO2013011061A1 (en) 2011-07-18 2013-01-24 Novo Nordisk A/S Antagonistic antibodies against oscar
JP2014521692A (en) 2011-08-04 2014-08-28 アムジエン・インコーポレーテツド A method of treating a bone gap defect
US9717803B2 (en) 2011-12-23 2017-08-01 Innate Pharma Enzymatic conjugation of polypeptides
CA2858974A1 (en) 2011-12-28 2013-07-04 Amgen Inc. Method of treating alveolar bone loss through the use of anti-sclerostin antibodies
ES2640268T3 (en) 2012-02-15 2017-11-02 Novo Nordisk A/S Antibodies which bind to a trigger block receptor expressed on myeloid cells 1 (TREM-1)
US9663568B2 (en) 2012-02-15 2017-05-30 Novo Nordisk A/S Antibodies that bind peptidoglycan recognition protein 1
EP2847219A1 (en) 2012-05-07 2015-03-18 Amgen Inc. Anti-erythropoietin antibodies
KR20150029678A (en) 2012-06-06 2015-03-18 바이오노르 이뮤노 에이에스 Vaccine
EP2869844A1 (en) 2012-07-05 2015-05-13 UCB Pharma S.A. Treatment for bone diseases
WO2014006230A1 (en) 2012-07-06 2014-01-09 Novo Nordisk A/S Il-20 epitopes and il-20 ligands
EP2872894A2 (en) 2012-07-13 2015-05-20 Innate Pharma Screening of conjugated antibodies
WO2014015217A1 (en) 2012-07-19 2014-01-23 Vertex Pharmaceuticals Incorporated Biomarkers for hcv infected patients
WO2014052672A1 (en) 2012-09-27 2014-04-03 Biocare Medical, Llc Anti-uroplakin ii antibodies systems and methods
CN104968677A (en) 2012-11-21 2015-10-07 安进公司 Heterodimeric immunoglobulins
US20140193410A1 (en) 2013-01-09 2014-07-10 University Of Miami Compositions and Methods for the Regulation of T Regulatory Cells Using TL1A-Ig Fusion Protein
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
EP2943511A4 (en) 2013-01-14 2016-08-31 Xencor Inc Novel heterodimeric proteins
US9738722B2 (en) 2013-01-15 2017-08-22 Xencor, Inc. Rapid clearance of antigen complexes using novel antibodies
EP2962113A4 (en) 2013-02-28 2016-11-09 Biocare Medical Llc Anti-p40 antibodies systems and methods
EP2962100A1 (en) 2013-02-28 2016-01-06 Caprion Proteomics Inc. Tuberculosis biomarkers and uses thereof
US20160032012A1 (en) 2013-03-15 2016-02-04 Novo Nordisk A/S Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
US9708375B2 (en) 2013-03-15 2017-07-18 Amgen Inc. Inhibitory polypeptides specific to WNT inhibitors
KR20160042871A (en) 2013-06-21 2016-04-20 이나뜨 파르마, 에스.아. Enzymatic conjugation of polypeptides
WO2015007337A1 (en) 2013-07-19 2015-01-22 Bionor Immuno As Method for the vaccination against hiv
CA2924172A1 (en) 2013-09-13 2015-03-19 Beigene, Ltd. Anti-pd1 antibodies and their use as therapeutics and diagnostics
WO2015051320A3 (en) 2013-10-03 2015-06-18 Biocare Medical, Llc Anti-sox10 antibody systems and methods
US20160297892A1 (en) 2013-11-07 2016-10-13 Novo Nordisk A/S Novel Methods and Antibodies for Treating Coagulapathy
CN106471009A (en) 2014-03-28 2017-03-01 Xencor公司 Bispecific antibodies that bind to CD38 and CD3
WO2016207273A3 (en) 2015-06-23 2017-02-16 Innate Pharma Multispecific antigen binding proteins
US20160369002A1 (en) 2015-06-23 2016-12-22 Innate Pharma Multispecific nk engager proteins
EP2975056A1 (en) 2014-07-17 2016-01-20 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US20160355608A1 (en) 2014-11-26 2016-12-08 Xencor, Inc. Heterodimeric antibodies that bind cd3 and tumor antigens
EP3026061A1 (en) 2014-11-26 2016-06-01 Novo Nordisk A/S Site directed mutagenesis of trem-1 antibodies for decreasing viscosity.
WO2016086189A3 (en) 2014-11-26 2016-09-09 Xencor, Inc. Heterodimeric antibodies that bind cd3 and tumor antigens
US20170340591A1 (en) 2014-12-23 2017-11-30 Inserm (Institut National De La Santé Et La Recherche Médicale Alpha-cell re-generation combined with conversion to beta cells

Citations (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4704692A (en) * 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4906562A (en) * 1984-12-21 1990-03-06 Oncogen Monocolonal antibodies and antigen for human non-small cell lung carcinomas
US4935495A (en) * 1984-12-21 1990-06-19 Oncogen Monoclonal antibodies to the L6 glycolipid antigenic determinant found on human non-small cell lung carcinomas
US4946778A (en) * 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5098833A (en) * 1989-02-23 1992-03-24 Genentech, Inc. DNA sequence encoding a functional domain of a lymphocyte homing receptor
US5225539A (en) * 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5260203A (en) * 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5434131A (en) * 1991-06-27 1995-07-18 Bristol Myers Squibb Co. Chimeric CTLA4 receptor and methods for its use
US5500362A (en) * 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5521288A (en) * 1990-03-26 1996-05-28 Bristol-Myers Squibb Company CD28IG fusion protein
US5605690A (en) * 1989-09-05 1997-02-25 Immunex Corporation Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor
US5637481A (en) * 1993-02-01 1997-06-10 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US5677425A (en) * 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US5709859A (en) * 1991-01-24 1998-01-20 Bristol-Myers Squibb Company Mixed specificity fusion proteins
US5714147A (en) * 1989-02-23 1998-02-03 Genentech Inc. Hybrid immunoglobulins
US5736137A (en) * 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5770197A (en) * 1991-06-27 1998-06-23 Bristol-Myers Squibb Company Methods for regulating the immune response using B7 binding molecules and IL4-binding molecules
US5773253A (en) * 1993-01-22 1998-06-30 Bristol-Myers Squibb Company MYPPPY variants of CTL A4 and uses thereof
US5776456A (en) * 1992-11-13 1998-07-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5844095A (en) * 1991-06-27 1998-12-01 Bristol-Myers Squibb Company CTLA4 Ig fusion proteins
US5844093A (en) * 1994-03-17 1998-12-01 Merck Patent Gesellschaft Mit Beschrankter Haftung Anti-EGFR single-chain Fvs and anti-EGFR antibodies
US5869049A (en) * 1993-09-02 1999-02-09 Trustees Of Dartmouth College Methods of inducing T cell unresponsiveness to bone marrow with gp39 antagonists
US5876718A (en) * 1993-09-02 1999-03-02 Trustees Of Dartmouth College Methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host-disease with anti-gp39 antibodies
US5916560A (en) * 1996-03-20 1999-06-29 Bristol-Myers Squibb Company Methods for inhibiting an immune response by blocking the GP39/CD40 and CTLA4/CD28/B7 pathways and compositions for use therewith
US6074644A (en) * 1995-10-13 2000-06-13 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acids encoding immunotoxins containing a disulfide-stabilized antibody fragment replacing half or more of domain IB of pseudomonas exotoxin, and methods of use of the encoded immunotoxins
US6087329A (en) * 1991-10-25 2000-07-11 Immunex Corporation CD40 ligand polypeptide
US6090914A (en) * 1991-06-27 2000-07-18 Bristol-Myers Squibb Company CTLA4/CD28Ig hybrid fusion proteins and uses thereof
US6147203A (en) * 1993-06-14 2000-11-14 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
US6194551B1 (en) * 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6284536B1 (en) * 1998-04-20 2001-09-04 The Regents Of The University Of California Modified immunoglobin molecules and methods for use thereof
US20010041350A1 (en) * 1995-03-31 2001-11-15 Human Genome Sciences, Inc. Human DNA ligase III
US20020039557A1 (en) * 2000-06-20 2002-04-04 Christine White Treatment of B-cell associated diseases such as malignances and autoimmune diseases using a cold anti-CD20 antibody/radiolabeled anti-CD22 antibody combination
US6376459B1 (en) * 1992-02-14 2002-04-23 Bristol-Myers Squibb Company Inhibiting B cell activation with soluble CD40 or fusion proteins thereof
US6384198B1 (en) * 1993-05-25 2002-05-07 Bristol-Myers Sqibb Company Methods for inhibiting the production of HIV-1 retrovirus using monoclonal antibodies and Fv specific for CD2 antigen
US6410319B1 (en) * 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US6444792B1 (en) * 1996-02-02 2002-09-03 Repligen Corporation CTLA4-Cγ4 fusion proteins
US20020155604A1 (en) * 1998-02-19 2002-10-24 Jeffrey A. Ledbetter Compositions and methods for regulating lymphocyte activation
US6472510B1 (en) * 1992-02-14 2002-10-29 Bristol-Myers Squibb Company CD40 receptor ligands
US6482919B2 (en) * 1993-02-01 2002-11-19 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US20030133930A1 (en) * 1999-06-09 2003-07-17 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20030219436A1 (en) * 2002-03-15 2003-11-27 Ledbetter Jeffrey A. Compositions and methods to regulate an immune response using CD83 gene expressed in tumors and using soluble CD83-Ig fusion protein
US20030219433A1 (en) * 2002-02-14 2003-11-27 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US20030219446A1 (en) * 1990-03-26 2003-11-27 Bristol-Myers Squibb Company Ligand for CD28 receptor on B cells and methods
US20040058445A1 (en) * 2001-04-26 2004-03-25 Ledbetter Jeffrey Alan Activation of tumor-reactive lymphocytes via antibodies or genes recognizing CD3 or 4-1BB
US7148321B2 (en) * 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety

Family Cites Families (150)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US219433A (en) * 1879-09-09 Improvement in grain-binders
US219436A (en) * 1879-09-09 Improvement in button-gaiters
US39557A (en) * 1863-08-18 Improvement in spindles of spinning-machines
US219446A (en) * 1879-09-09 Improvement in electric telephones
US219876A (en) * 1879-09-23 Improvement in calk attachments
US44135A (en) * 1864-09-06 Improved sorghum-evaporator
US31510A (en) * 1861-02-19 atwood
US133930A (en) * 1872-12-17 Improvement in toy spring-guns
US58445A (en) * 1866-10-02 Improvement in ties for cotton-bales
US588773A (en) * 1897-08-24 Umbrella or parasol
US44423A (en) * 1864-09-27 Improved screw for music-stools
US155604A (en) * 1874-10-06 Improvement in secretaries
US6893625B1 (en) 1986-10-27 2005-05-17 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US5955315A (en) 1985-11-19 1999-09-21 Schering Corporation Nucleic acids encoding human interleukin-4
US6027725A (en) 1991-11-25 2000-02-22 Enzon, Inc. Multivalent antigen-binding proteins
US5869620A (en) * 1986-09-02 1999-02-09 Enzon, Inc. Multivalent antigen-binding proteins
US5892019A (en) 1987-07-15 1999-04-06 The United States Of America, As Represented By The Department Of Health And Human Services Production of a single-gene-encoded immunoglobulin
GB8720833D0 (en) 1987-09-04 1987-10-14 Celltech Ltd Recombinant dna product
WO1989007142A1 (en) 1988-02-05 1989-08-10 Morrison Sherie L Domain-modified constant region antibodies
WO1989008114A1 (en) 1988-02-25 1989-09-08 The General Hospital Corporation Rapid immunoselection cloning method
US5506126A (en) 1988-02-25 1996-04-09 The General Hospital Corporation Rapid immunoselection cloning method
US4861579A (en) 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO1991004329A1 (en) 1989-09-20 1991-04-04 Abbott Laboratories Method of producing fusion proteins
US6150584A (en) * 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5314995A (en) 1990-01-22 1994-05-24 Oncogen Therapeutic interleukin-2-antibody based fusion proteins
JPH07507440A (en) 1990-03-02 1995-08-24
GB9009106D0 (en) 1990-04-23 1990-06-20 3I Res Expl Ltd Processes and intermediates for synthetic antibody derivatives
EP0537293A4 (en) 1990-07-02 1993-09-08 Bristol-Myers Company Ligand for cd28 receptor on b cells and methods
DE69226877T2 (en) 1991-05-31 1999-03-11 Genentech Inc Treatment of HIV-associated immune thrombocytopenia purpura
DE69327620D1 (en) 1992-08-18 2000-02-24 Immunologia Molecular Ciudad D Monoclonal antibodies against the epidermal growth factor receptor, cells and methods for their preparation and preserving compositions
DE69334258D1 (en) 1992-08-21 2009-02-26 Univ Bruxelles Immunoglobulins without light chain
CA2143417A1 (en) 1992-09-09 1994-03-17 Mitchell S. Gross Novel antibodies for conferring passive immunity against infection by a pathogen in humans
US5597707A (en) * 1993-04-15 1997-01-28 Bristol-Myers Squibb Company Tumor associated antigen recognized by the murine monoclonal antibody L6, its oligonucleotide sequence and methods for their use
DE69427974T2 (en) 1993-04-29 2001-12-06 Unilever Nv Preparation made of antibodies or functional parts thereof derived from heavy chains of immunoglobulins of camelidae
US5417972A (en) 1993-08-02 1995-05-23 The Board Of Trustees Of The Leland Stanford Junior University Method of killing B-cells in a complement independent and an ADCC independent manner using antibodies which specifically bind CDIM
US5595721A (en) 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
WO1995009917A1 (en) 1993-10-07 1995-04-13 The Regents Of The University Of California Genetically engineered bispecific tetravalent antibodies
CA2182498A1 (en) 1994-02-01 1995-08-10 William J. Larochelle Fusion proteins that include antibody and nonantibody portions
JPH07226395A (en) * 1994-02-15 1995-08-22 Matsushita Electric Ind Co Ltd Vacuum plasma treatment apparatus
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
US5888773A (en) 1994-08-17 1999-03-30 The United States Of America As Represented By The Department Of Health And Human Services Method of producing single-chain Fv molecules
US5876950A (en) * 1995-01-26 1999-03-02 Bristol-Myers Squibb Company Monoclonal antibodies specific for different epitopes of human GP39 and methods for their use in diagnosis and therapy
EP0739981A1 (en) 1995-04-25 1996-10-30 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
US6440418B1 (en) 1995-11-07 2002-08-27 Idec Pharmaceuticals Corporation Methods of treating autoimmune diseases with gp39-specific antibodies
GB9604769D0 (en) 1996-03-06 1996-05-08 Efford Clive W Multi drive unit
EP1186300A1 (en) 1996-03-20 2002-03-13 Bristol-Myers Squibb Company Pharmaceutical compositions useful for inhibiting an immune response by blocking the GP39/CD40 and CTLA4/CD28/B7 pathways
EP0938556B1 (en) 1996-07-12 2005-03-09 Genentech, Inc. Gamma-heregulin
DE69726003T2 (en) 1996-07-16 2004-08-26 Andreas Plückthun Immunoglobulin superfamily domains and fragments with increased solubility
US5972361A (en) 1996-10-25 1999-10-26 The Procter & Gamble Company Cleansing products
US20020041847A1 (en) 1998-03-12 2002-04-11 Goldenberg David M. Immunotherapy of malignant and autoimmune disorders in domestic animals using naked antibodies, immunoconjugates and fusion proteins
US6306393B1 (en) 1997-03-24 2001-10-23 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
ES2190087T3 (en) 1997-06-13 2003-07-16 Genentech Inc Stabilized formulation of an antibody.
CA2293829C (en) 1997-06-24 2011-06-14 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US5994511A (en) 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
US6368596B1 (en) 1997-07-08 2002-04-09 Board Of Regents, The University Of Texas System Compositions and methods for homoconjugates of antibodies which induce growth arrest or apoptosis of tumor cells
CA2307166A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
DE19751651C1 (en) * 1997-11-21 1999-02-18 Bosch Gmbh Robert Circuit for switching an inductive load
CA2320403A1 (en) 1998-02-25 1999-09-02 Lexigen Pharmaceuticals Corporation Enhancing the circulating half-life of antibody-based fusion proteins
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
JP2002510481A (en) 1998-04-02 2002-04-09 ジェネンテック・インコーポレーテッド Antibody variants and fragments thereof
GB9809280D0 (en) 1998-04-30 1998-07-01 Rpms Technology Ltd Immunosupression
WO1999061630A3 (en) * 1998-05-26 2000-01-27 Regeneron Pharma Chimeric molecules comprising an extracellular ligand binding domain of a receptor and an ige fc or constant region, and their use in an assay system
KR20080038453A (en) 1998-08-11 2008-05-06 바이오겐 아이덱 인크. A pharmaceutical product comprising an anti-cd20 antibody to treat relapsed b-cell lymphoma
US6224866B1 (en) 1998-10-07 2001-05-01 Biocrystal Ltd. Immunotherapy of B cell involvement in progression of solid, nonlymphoid tumors
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2000027885A1 (en) 1998-11-05 2000-05-18 Kyowa Hakko Kogyo Co., Ltd. Novel chimeric polypeptide
DE69941903D1 (en) 1998-11-09 2010-02-25 Biogen Idec Inc Treatment of patients, the blood stem cells, a bone marrow transplant or a transplant of peripheral obtained with anti-CD20 ab
CN1679934B (en) 1998-11-09 2012-09-05 生物基因Idec公司 Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-CD20 antibody
US6150594A (en) * 1998-11-14 2000-11-21 The Mike Fiondella School Of Drumming L.L.C. Drumming apparatus
KR101155191B1 (en) 1999-01-15 2012-06-13 제넨테크, 인크. Polypeptide Variants with Altered Effector Function
US6897044B1 (en) 1999-01-28 2005-05-24 Biogen Idec, Inc. Production of tetravalent antibodies
CN1345334A (en) 1999-01-29 2002-04-17 伊姆克罗尼系统公司 Antibodies specific to KDR and uses thereof
US6383276B1 (en) 1999-03-12 2002-05-07 Fuji Photo Film Co., Ltd. Azomethine compound and oily magenta ink
GB9909925D0 (en) 1999-04-29 1999-06-30 Pharmacia & Upjohn Spa Combined preparations comprising anthracycline derivatives
WO2000067796A1 (en) 1999-05-07 2000-11-16 Genentech, Inc. Treatment of autoimmune diseases with antagonists which bind to b cell surface markers
JP2000353767A (en) * 1999-05-14 2000-12-19 Universal Instr Corp Board for mounting electronic component, package, mounting method, and method for housing integrated circuit chip in package
WO2000069913A1 (en) 1999-05-19 2000-11-23 Lexigen Pharmaceuticals Corp. EXPRESSION AND EXPORT OF INTERFERON-ALPHA PROTEINS AS Fc FUSION PROTEINS
CA2376288A1 (en) 1999-06-11 2000-12-21 Consiglio Nazionale Delle Ricerche Use of antibodies against cd20 for the treatment of the graft versus host disease
DE19930748C2 (en) 1999-07-02 2001-05-17 Infineon Technologies Ag A process for the manufacture of EEPROM and DRAM grave memory cell regions on a chip
EP1216056A1 (en) 1999-07-12 2002-06-26 Genentech Inc. Blocking immune response to a foreign antigen using an antagonist which binds to cd20
CN101259270A (en) 1999-08-11 2008-09-10 拜奥根Idec公司 Treatment of patients having non-hodgkins lymphoma with bone marrow involvement with anti-CD20 antibodies
US6451284B1 (en) 1999-08-11 2002-09-17 Idec Pharmaceuticals Corporation Clinical parameters for determining hematologic toxicity prior to radioimmunotheraphy
US8557244B1 (en) 1999-08-11 2013-10-15 Biogen Idec Inc. Treatment of aggressive non-Hodgkins lymphoma with anti-CD20 antibody
WO2001013945A1 (en) 1999-08-23 2001-03-01 Biocrystal Ltd. Methods and compositions for immunotherapy of b cell involvement in promotion of a disease condition comprising multiple sclerosis
KR20080039547A (en) 2000-07-12 2008-05-07 아이덱 파마슈티칼즈 코포레이션 Treatment of b cell malignancies using combination of b cell depleting antibody and immune modulating antibody related applications
US20020006404A1 (en) 1999-11-08 2002-01-17 Idec Pharmaceuticals Corporation Treatment of cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US20020009427A1 (en) 2000-03-24 2002-01-24 Wolin Maurice J. Methods of therapy for non-hodgkin's lymphoma
CA2404365A1 (en) 2000-03-31 2001-10-11 Idec Pharmaceutical Corporation Combined use of anti-cytokine antibodies or antagonists and anti-cd20 for the treatment of b cell lymphoma
KR20020093029A (en) 2000-04-11 2002-12-12 제넨테크, 인크. Multivalent Antibodies And Uses Therefor
ES2527754T3 (en) 2000-04-25 2015-01-29 Icos Corporation Delta isoform inhibitors of phosphatidylinositol 3-kinase human
EP1939203B1 (en) 2000-04-25 2014-11-19 ICOS Corporation Inhibitors of human phosphatidyl-inositol 3-kinase delta isoform
EP1286692A4 (en) 2000-04-25 2004-11-17 Idec Pharma Corp Intrathecal administration of rituximab for treatment of central nervous system lymphomas
US6667300B2 (en) 2000-04-25 2003-12-23 Icos Corporation Inhibitors of human phosphatidylinositol 3-kinase delta
DK1296714T3 (en) 2000-06-22 2009-12-07 Coley Pharm Gmbh Combination of CpG and antibodies directed against CD19, CD20, CD22 or CD40 for the treatment or prevention of cancer
US20020028178A1 (en) * 2000-07-12 2002-03-07 Nabil Hanna Treatment of B cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
WO2002034790A1 (en) 2000-10-20 2002-05-02 Idec Pharmaceuticals Corporation Variant igg3 rituxan r and therapeutic use thereof
US7754209B2 (en) 2003-07-26 2010-07-13 Trubion Pharmaceuticals Binding constructs and methods for use thereof
CN1911965B (en) 2001-01-17 2013-05-29 新兴产品开发西雅图有限公司 Binding domain-immunoglobulin fusion proteins
US7829084B2 (en) 2001-01-17 2010-11-09 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
EP1373321A2 (en) 2001-01-29 2004-01-02 Idec Pharmaceuticals Corporation Modified antibodies and methods of use
WO2002096948A3 (en) 2001-01-29 2003-08-07 Idec Pharma Corp Engineered tetravalent antibodies and methods of use
CA2440831C (en) 2001-04-02 2013-05-28 Genentech, Inc. Combination therapy using cd40 and cd20 ligands
JP2005500018A (en) 2001-04-02 2005-01-06 アイデック ファーマスーティカルズ コーポレイション GnTIII and co-expressing recombinant antibody
EP1404366A4 (en) 2001-06-14 2006-06-07 Intermune Inc Combination therapy of gamma-interferon and b cell specific antibodies
ES2263984T3 (en) 2002-06-28 2006-12-16 Domantis Limited Double-specific ligands with increased serum half-life.
EP1539233B1 (en) 2001-07-12 2011-04-27 FOOTE, Jefferson Super humanized antibodies
US6681649B2 (en) * 2002-04-03 2004-01-27 Honeywell International Inc. Inertial control and measurement system
WO2003106622A3 (en) 2002-05-30 2004-07-01 Valerie I Brown Methods for treatment of acute lymphocytic leukemia
US6869049B2 (en) * 2002-07-24 2005-03-22 General Electric Company Method and apparatus for modulating flow separation
EP1576182B1 (en) 2002-12-23 2010-11-17 Bristol-Myers Squibb Company Product quality enhancement in mammalian cell culture processes for protein production
WO2005087265A8 (en) 2004-03-11 2006-10-05 Lee F Allen Antineoplastic combinations of cci-779 and rituximab
US7393662B2 (en) 2004-09-03 2008-07-01 Centocor, Inc. Human EPO mimetic hinge core mimetibodies, compositions, methods and uses
KR101251157B1 (en) 2005-07-25 2013-04-10 이머전트 프로덕트 디벨롭먼트 시애틀, 엘엘씨 B-cell reduction using cd37-specific and cd20-specific binding molecules
US20080279850A1 (en) 2005-07-25 2008-11-13 Trubion Pharmaceuticals, Inc. B-Cell Reduction Using CD37-Specific and CD20-Specific Binding Molecules
RU2421242C2 (en) 2005-07-25 2011-06-20 Трабьон Фармасьютикалз, Инк. Application of single dose of cd20-specific binding molecules
WO2008053275A3 (en) 2005-12-20 2008-08-21 Morphosys Ag Novel collection of hcdr3 regions and uses therefor
WO2007146968A3 (en) 2006-06-12 2008-06-19 Trubion Pharmaceuticals Inc Single-chain multivalent binding proteins with effector function
WO2008153636A1 (en) 2007-05-04 2008-12-18 Cellsignaling Technology, Inc. Phospho-specific antibodies to p13k regulatory subunit and uses thereof
JP2010507394A (en) 2006-10-24 2010-03-11 トルービオン ファーマスーティカルズ インコーポレイテッド Materials and methods for improved immunoglycoprotein
US7846434B2 (en) 2006-10-24 2010-12-07 Trubion Pharmaceuticals, Inc. Materials and methods for improved immunoglycoproteins
JP2010526120A (en) 2007-05-09 2010-07-29 ノバルティス アーゲー Substituted imidazopyridazine as Pi3k lipid kinase inhibitors
WO2008152390A1 (en) 2007-06-12 2008-12-18 F.Hoffmann-La Roche Ag Thiazoliopyrimidines and their use as inhibitors of phosphatidylinositol-3 kinase
WO2008152394A1 (en) 2007-06-12 2008-12-18 F.Hoffmann-La Roche Ag Pharmaceutical compounds
WO2008152387A1 (en) 2007-06-12 2008-12-18 F.Hoffmann-La Roche Ag Quinazoline derivatives as pi3 kinase inhibitors
WO2009023386A3 (en) 2007-07-06 2009-11-19 Trubion Pharmaceuticals, Inc. Binding peptides having a c-terminally disposed specific binding domain
CA2693464C (en) 2007-08-09 2017-10-24 Boehringer Ingelheim International Gmbh Anti-cd37 antibodies
CA2699202C (en) 2007-09-12 2016-09-27 F. Hoffmann-La Roche Ag Combinations of phosphoinositide 3-kinase inhibitor compounds and chemotherapeutic agents, and methods of use
US20100204222A1 (en) 2007-09-17 2010-08-12 GlaxoSmithKline, LLC Pyridopyrimidine derivatives as p13 kinase inhibitors
CA2704711C (en) 2007-09-24 2016-07-05 Genentech, Inc. Thiazolopyrimidine p13k inhibitor compounds and methods of use
WO2009040552A3 (en) 2007-09-27 2009-05-22 Ct Nac De Investigaciones Onco Substituted imidazo (2, 1-b) -1, 3, 4-thiazole compounds, their pharmaceutical compositions and uses thereof
GB2467670B (en) 2007-10-04 2012-08-01 Intellikine Inc Chemical entities and therapeutic uses thereof
EP2209785A1 (en) 2007-10-05 2010-07-28 S*BIO Pte Ltd 2-morpholinylpurines as inhibitors of pi3k
DK2209786T3 (en) 2007-10-05 2013-06-03 Verastem Inc Pyrimidinsubstituerede purine derivatives
KR20100083170A (en) 2007-10-16 2010-07-21 와이어쓰 엘엘씨 Thienopyrimidine and pyrazolopyrimidine compounds and their use as mtor kinase and pi3 kinase inhibitors
WO2009055418A1 (en) 2007-10-22 2009-04-30 Smithkline Beecham Corporation Pyridosulfonamide derivatives as pi3 kinase inhibitors
EP2215090B1 (en) 2007-10-26 2015-03-04 F. Hoffmann-La Roche AG Purine derivatives useful as pi3 kinase inhibitors
GB0721095D0 (en) 2007-10-26 2007-12-05 Piramed Ltd Pharmaceutical compounds
US20090131512A1 (en) 2007-10-31 2009-05-21 Dynavax Technologies Corp. Inhibition of type I in IFN production
WO2009059030A1 (en) 2007-10-31 2009-05-07 Burnham Institute For Medical Research Pyrazole derivatives as kinase inhibitors
CN101952292A (en) 2007-11-13 2011-01-19 艾科斯有限公司 Inhibitors of human phosphatidyl-inositol 3-kinase delta
WO2009066084A1 (en) 2007-11-21 2009-05-28 F. Hoffmann-La Roche Ag 2 -morpholinopyrimidines and their use as pi3 kinase inhibitors
WO2009070524A1 (en) 2007-11-27 2009-06-04 Wyeth Pyrrolo[3,2-d]pyrimidine compounds and their use as pi3 kinase and mtor kinase inhibitors
CN101952285A (en) 2007-11-27 2011-01-19 塞尔卓姆有限公司 Amino triazoles as PI3K inhibitors
US8333966B2 (en) 2008-04-11 2012-12-18 Emergent Product Development Seattle, Llc CD37 immunotherapeutics and uses thereof
US20100135900A1 (en) 2008-11-13 2010-06-03 Trubion Pharmaceuticals, Inc. Cd37 immunotherapeutic combination therapies and uses thereof

Patent Citations (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4906562A (en) * 1984-12-21 1990-03-06 Oncogen Monocolonal antibodies and antigen for human non-small cell lung carcinomas
US4935495A (en) * 1984-12-21 1990-06-19 Oncogen Monoclonal antibodies to the L6 glycolipid antigenic determinant found on human non-small cell lung carcinomas
US5091177A (en) * 1984-12-21 1992-02-25 Oncogen Monoclonal antibodies for treatment of human non-small cell lung carcinomas
US5225539A (en) * 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5455030A (en) * 1986-09-02 1995-10-03 Enzon Labs, Inc. Immunotheraphy using single chain polypeptide binding molecules
US4704692A (en) * 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5260203A (en) * 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5500362A (en) * 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5677425A (en) * 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US4946778A (en) * 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5098833A (en) * 1989-02-23 1992-03-24 Genentech, Inc. DNA sequence encoding a functional domain of a lymphocyte homing receptor
US5714147A (en) * 1989-02-23 1998-02-03 Genentech Inc. Hybrid immunoglobulins
US5605690A (en) * 1989-09-05 1997-02-25 Immunex Corporation Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor
US5521288A (en) * 1990-03-26 1996-05-28 Bristol-Myers Squibb Company CD28IG fusion protein
US5580756A (en) * 1990-03-26 1996-12-03 Bristol-Myers Squibb Co. B7Ig fusion protein
US20030219446A1 (en) * 1990-03-26 2003-11-27 Bristol-Myers Squibb Company Ligand for CD28 receptor on B cells and methods
US6641809B1 (en) * 1990-03-26 2003-11-04 Bristol-Myers Squibb Company Method of regulating cellular processes mediated by B7 and CD28
US5709859A (en) * 1991-01-24 1998-01-20 Bristol-Myers Squibb Company Mixed specificity fusion proteins
US5434131A (en) * 1991-06-27 1995-07-18 Bristol Myers Squibb Co. Chimeric CTLA4 receptor and methods for its use
US5770197A (en) * 1991-06-27 1998-06-23 Bristol-Myers Squibb Company Methods for regulating the immune response using B7 binding molecules and IL4-binding molecules
US6623940B1 (en) * 1991-06-27 2003-09-23 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US5844095A (en) * 1991-06-27 1998-12-01 Bristol-Myers Squibb Company CTLA4 Ig fusion proteins
US6090914A (en) * 1991-06-27 2000-07-18 Bristol-Myers Squibb Company CTLA4/CD28Ig hybrid fusion proteins and uses thereof
US20030219876A1 (en) * 1991-06-27 2003-11-27 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US6264951B1 (en) * 1991-10-25 2001-07-24 Immunex Corporation Methods of inhibiting CD40L binding to CD40 with soluble monomeric CD40L
US6087329A (en) * 1991-10-25 2000-07-11 Immunex Corporation CD40 ligand polypeptide
US6472510B1 (en) * 1992-02-14 2002-10-29 Bristol-Myers Squibb Company CD40 receptor ligands
US6376459B1 (en) * 1992-02-14 2002-04-23 Bristol-Myers Squibb Company Inhibiting B cell activation with soluble CD40 or fusion proteins thereof
US5776456A (en) * 1992-11-13 1998-07-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5736137A (en) * 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5773253A (en) * 1993-01-22 1998-06-30 Bristol-Myers Squibb Company MYPPPY variants of CTL A4 and uses thereof
US6482919B2 (en) * 1993-02-01 2002-11-19 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US5637481A (en) * 1993-02-01 1997-06-10 Bristol-Myers Squibb Company Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US6384198B1 (en) * 1993-05-25 2002-05-07 Bristol-Myers Sqibb Company Methods for inhibiting the production of HIV-1 retrovirus using monoclonal antibodies and Fv specific for CD2 antigen
US6147203A (en) * 1993-06-14 2000-11-14 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
US5876718A (en) * 1993-09-02 1999-03-02 Trustees Of Dartmouth College Methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host-disease with anti-gp39 antibodies
US6312692B1 (en) * 1993-09-02 2001-11-06 Trustees Of Dartmouth College Method of treating graft-versus-host disease with anti-GP39 antibodies and bone marrow cells
US5869049A (en) * 1993-09-02 1999-02-09 Trustees Of Dartmouth College Methods of inducing T cell unresponsiveness to bone marrow with gp39 antagonists
US5844093A (en) * 1994-03-17 1998-12-01 Merck Patent Gesellschaft Mit Beschrankter Haftung Anti-EGFR single-chain Fvs and anti-EGFR antibodies
US20010041350A1 (en) * 1995-03-31 2001-11-15 Human Genome Sciences, Inc. Human DNA ligase III
US6074644A (en) * 1995-10-13 2000-06-13 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acids encoding immunotoxins containing a disulfide-stabilized antibody fragment replacing half or more of domain IB of pseudomonas exotoxin, and methods of use of the encoded immunotoxins
US6444792B1 (en) * 1996-02-02 2002-09-03 Repligen Corporation CTLA4-Cγ4 fusion proteins
US20020031510A1 (en) * 1996-03-20 2002-03-14 Bristol-Myers Squibb Company Methods for inhibiting an immune response by blocking the GP39/CD40 and CTLA4/CD28/B7 pathways and compositions for use therewith
US5916560A (en) * 1996-03-20 1999-06-29 Bristol-Myers Squibb Company Methods for inhibiting an immune response by blocking the GP39/CD40 and CTLA4/CD28/B7 pathways and compositions for use therewith
US20020155604A1 (en) * 1998-02-19 2002-10-24 Jeffrey A. Ledbetter Compositions and methods for regulating lymphocyte activation
US6194551B1 (en) * 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6284536B1 (en) * 1998-04-20 2001-09-04 The Regents Of The University Of California Modified immunoglobin molecules and methods for use thereof
US6410319B1 (en) * 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US20030133930A1 (en) * 1999-06-09 2003-07-17 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20020039557A1 (en) * 2000-06-20 2002-04-04 Christine White Treatment of B-cell associated diseases such as malignances and autoimmune diseases using a cold anti-CD20 antibody/radiolabeled anti-CD22 antibody combination
US7148321B2 (en) * 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US20040058445A1 (en) * 2001-04-26 2004-03-25 Ledbetter Jeffrey Alan Activation of tumor-reactive lymphocytes via antibodies or genes recognizing CD3 or 4-1BB
US20030219433A1 (en) * 2002-02-14 2003-11-27 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US20030219436A1 (en) * 2002-03-15 2003-11-27 Ledbetter Jeffrey A. Compositions and methods to regulate an immune response using CD83 gene expressed in tumors and using soluble CD83-Ig fusion protein

Cited By (445)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8106161B2 (en) 2001-01-17 2012-01-31 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US20030118592A1 (en) * 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US9005612B2 (en) 2001-01-17 2015-04-14 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8197810B2 (en) 2001-01-17 2012-06-12 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8147835B2 (en) 2001-01-17 2012-04-03 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US7754208B2 (en) * 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US8188237B2 (en) 2001-01-17 2012-05-29 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US20100034820A1 (en) * 2001-01-17 2010-02-11 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050202028A1 (en) * 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20110105729A1 (en) * 2001-01-17 2011-05-05 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20110091461A1 (en) * 2001-01-17 2011-04-21 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20110223164A1 (en) * 2001-01-17 2011-09-15 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US20060263856A1 (en) * 2001-03-07 2006-11-23 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US8066994B2 (en) 2001-03-07 2011-11-29 Merck Patent Gmbh Proteins comprising an IgG2 domain
US20040058445A1 (en) * 2001-04-26 2004-03-25 Ledbetter Jeffrey Alan Activation of tumor-reactive lymphocytes via antibodies or genes recognizing CD3 or 4-1BB
US20070212349A1 (en) * 2001-10-16 2007-09-13 Government Of The United States Of America, Represented By The Secretary, Department Broadly cross-reactive neutralizing antibodies against human immunodeficiency virus selected by env-cd4-co-receptor complexes
US7378093B2 (en) 2001-10-16 2008-05-27 The United States Of America As Represented By The Department Of Health And Human Services Broadly cross-reactive neutralizing antibodies against Human Immunodeficiency Virus selected by Env-CD4-co-receptor complexes
US20070160597A1 (en) * 2002-03-01 2007-07-12 Xencor, Inc. Optimized Fc variants and methods for their generation
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US20080260731A1 (en) * 2002-03-01 2008-10-23 Bernett Matthew J Optimized antibodies that target cd19
US20070224189A1 (en) * 2002-03-01 2007-09-27 Xencor, Inc. CD20 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
US7662925B2 (en) 2002-03-01 2010-02-16 Xencor, Inc. Optimized Fc variants and methods for their generation
US20080254027A1 (en) * 2002-03-01 2008-10-16 Bernett Matthew J Optimized CD5 antibodies and methods of using the same
US20070003546A1 (en) * 2002-03-01 2007-01-04 Xencor, Inc. Optimized Fc variants and methods for their generation
US20070219133A1 (en) * 2002-03-01 2007-09-20 Xencor, Inc. CD52 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
US20090142340A1 (en) * 2002-03-01 2009-06-04 Xencor, Inc. Optimized Fc Variants and Methods for Their Generation
US20080181890A1 (en) * 2002-03-01 2008-07-31 Xencor, Inc. Optimized Fc Variants and Methods for Their Generation
US8093357B2 (en) 2002-03-01 2012-01-10 Xencor, Inc. Optimized Fc variants and methods for their generation
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
US8124731B2 (en) 2002-03-01 2012-02-28 Xencor, Inc. Optimized Fc variants and methods for their generation
US20090068175A1 (en) * 2002-03-01 2009-03-12 Xencor, Inc. Optimized FC Variants and Methods for Their Generation
US20030219436A1 (en) * 2002-03-15 2003-11-27 Ledbetter Jeffrey A. Compositions and methods to regulate an immune response using CD83 gene expressed in tumors and using soluble CD83-Ig fusion protein
US8110192B2 (en) 2002-05-06 2012-02-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human immunodeficiency virus type 1 (HIV-1)-neutralizing human single-chain antibodies with improved breadth and potency
US20050123900A1 (en) * 2002-05-06 2005-06-09 Dimitrov Dimiter S. Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries
US7803913B2 (en) 2002-05-06 2010-09-28 The United States Of America As Represented By The Department Of Health And Human Services Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries
US20090291493A1 (en) * 2002-05-06 2009-11-26 The U.S.A, As Represented By The Secretary, Department Of Health And Human Services Human immunodeficiency virus-neutralizing antibodies with improved breadth and potency
US20050164166A1 (en) * 2002-05-06 2005-07-28 Government Of The United States Of America Represented By The Secreatary, Dept Of Health Serv Identification of novel broadly cross-reactive hiv-1 neutralizing human monoclonal Antibodies
US7566451B2 (en) 2002-05-06 2009-07-28 The United States Of America As Represented By The Department Of Health And Human Services Human immunodeficiency virus-neutralizing human antibodies with improved breadth and potency
US20100317837A1 (en) * 2002-05-06 2010-12-16 The United States of America, as represented by the Secretary, Dep. of Health and Human Services Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries
US9353187B2 (en) 2002-09-27 2016-05-31 Xencor, Inc. Optimized FC variants and methods for their generation
US8802823B2 (en) 2002-09-27 2014-08-12 Xencor, Inc. Optimized Fc variants
US8753629B2 (en) 2002-09-27 2014-06-17 Xencor, Inc. Optimized Fc variants
US20090081208A1 (en) * 2002-09-27 2009-03-26 Xencor, Inc. Optimized Fc variants and methods for their generation
US20040132101A1 (en) * 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US8093359B2 (en) 2002-09-27 2012-01-10 Xencor, Inc. Optimized Fc variants and methods for their generation
US20090092599A1 (en) * 2002-09-27 2009-04-09 Xencor, Inc. Optimized Fc variants and methods for their generation
US8039592B2 (en) 2002-09-27 2011-10-18 Xencor, Inc. Optimized Fc variants and methods for their generation
US20070148171A1 (en) * 2002-09-27 2007-06-28 Xencor, Inc. Optimized anti-CD30 antibodies
US8809503B2 (en) 2002-09-27 2014-08-19 Xencor, Inc. Optimized Fc variants and methods for their generation
US8858937B2 (en) 2002-09-27 2014-10-14 Xencor, Inc. Optimized Fc variants and methods for their generation
US8383109B2 (en) 2002-09-27 2013-02-26 Xencor, Inc. Optimized Fc variants and methods for their generation
US20060235208A1 (en) * 2002-09-27 2006-10-19 Xencor, Inc. Fc variants with optimized properties
US9193798B2 (en) 2002-09-27 2015-11-24 Xencor, Inc. Optimized Fc variants and methods for their generation
US8735547B2 (en) 2002-09-27 2014-05-27 Xencor, Inc. Optimized Fc Variants
US8188231B2 (en) 2002-09-27 2012-05-29 Xencor, Inc. Optimized FC variants
US8753628B2 (en) 2002-09-27 2014-06-17 Xencor, Inc. Optimized Fc variants
EP2316856A1 (en) 2002-10-17 2011-05-04 Genmab A/S Human monoclonal antibodies against CD20
EP2330130A1 (en) 2002-10-17 2011-06-08 Genmab A/S Human monoclonal antibodies against CD20
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US20060104971A1 (en) * 2002-12-20 2006-05-18 Biogen Idec Ma Inc. Multivalent lymphotoxin beta receptor agonists and therapeutic uses thereof
US8735545B2 (en) 2003-03-03 2014-05-27 Xencor, Inc. Fc variants having increased affinity for fcyrllc
US20070275460A1 (en) * 2003-03-03 2007-11-29 Xencor.Inc. Fc Variants With Optimized Fc Receptor Binding Properties
US8388955B2 (en) 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US20080057056A1 (en) * 2003-03-03 2008-03-06 Xencor, Inc. Fc Variants with Increased Affinity for FcyRIIC
US20070248603A1 (en) * 2003-03-03 2007-10-25 Xencor, Inc. Fc Variants with Increased Affinity for FcyRlla
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US20070248602A1 (en) * 2003-03-03 2007-10-25 Xencor, Inc. Fc Variants Having Increased Affinity for FcyRllc
US20070238665A1 (en) * 2003-03-03 2007-10-11 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIc
US20070243188A1 (en) * 2003-03-03 2007-10-18 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRlla
US20070237766A1 (en) * 2003-03-03 2007-10-11 Xencor, Inc. Fc Variants Having Increased Affinity for FcyRllla
US20070237765A1 (en) * 2003-03-03 2007-10-11 Xencor, Inc. Fc Variants Having Increased Affinity for FcyRl
US20110021755A1 (en) * 2003-03-03 2011-01-27 Xencor, Inc. Optimized Fc Variants
US20090010920A1 (en) * 2003-03-03 2009-01-08 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIb
US9663582B2 (en) 2003-03-03 2017-05-30 Xencor, Inc. Optimized Fc variants
US20070237767A1 (en) * 2003-03-03 2007-10-11 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRllla
US9657106B2 (en) 2003-03-03 2017-05-23 Xencor, Inc. Optimized Fc variants
US9051373B2 (en) 2003-05-02 2015-06-09 Xencor, Inc. Optimized Fc variants
JP2007528194A (en) * 2003-07-26 2007-10-11 トルビオン・ファーマシューティカルズ・インコーポレイテッドTrubion Pharmaceuticals, Inc. Binding constructs and methods of use thereof
JP4904443B2 (en) * 2003-07-26 2012-03-28 エマージェント プロダクト デベロップメント シアトル, エルエルシー Binding constructs and methods of use thereof